Chemiluminescent acridinum compounds and analogues thereof as substrates of hydrolytic enzymes

ABSTRACT

A chemiluminescent substrate of a hydrolytic enzyme having the following general Formula I is disclosed, as follows: 
 
Lumi-M-P  Formula I 
 
where “Lumi” is a chemiluminescent moiety capable of producing light (a) by itself, (b) with MP attached and (c) with M attached. Examples of Lumi include chemiluminescent acridinium compounds, benzacridinium compounds, quinolinium compounds, isoquinolinium compounds, phenanthridinium compounds, and lucigenin compounds, spiroacridan compounds, luminol compounds and isoluminol compounds. M is a multivalent heteroatom having at least one lone pair of electrons selected from oxygen, nitrogen and sulfur, directly attached to the light emitting moiety of Lumi at one end and to P at the other end. P is a group that can be readily removed by hydrolytic enzymes. An enzymatic reaction utilizing the above compound is the following:  
                 
where HE is a hydrolytic enzyme. Lumi-M is a chemiluminescent product having physical and/or chemical properties different from those of Lumi-M-P.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/626,566 filed on Jul. 27, 2000 and further claims priority of U.S.Provisional Application No. 60/146,648, filed Jul. 30, 1999, both ofwhich are hereby incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

N/A

FIELD OF THE INVENTION

This invention relates to novel chemiluminescent compounds that aresubstrates of hydrolytic enzymes, the chemiluminescent products of whichhave distinctly different light emission characteristics (i.e., emissionwavelength, kinetics, or quantum yield). This invention also relates toa light-releasing reagent composition that reacts preferentially withthe chemiluminescent substrate or with the chemiluminescent product inthe mixture of the two, to generate a discernible signal that can bequantified. This invention further relates to detection methodscomprising a novel acridinium-based chemiluminescent substrate, ahydrolytic enzyme and a signal-releasing reagent. This inventionfurthermore relates to detection devices in conjunction with the use ofnovel chemiluminescent substrates and hydrolytic enzymes, which includea red-sensitive photomultiplier tube or a charge-coupled device, and alight-filtering device to maximize the detection of chemiluminescentproduct signal. Further, this invention relates to the use of thesenovel chemiluminescent substrates in assays to detect, quantitatively orqualitatively, a hydrolytic enzyme of interest that is present either asa label or as a marker of a biological sample. Finally, this inventionrelates to the process and intermediates for the preparation of thesenovel chemiluminescent substrates.

BACKGROUND OF THE INVENTION

The detection of hydrolytic enzymes has been extensively used indiagnostic assays ranging from immunoassays, nucleic acid assays,receptor assays, and other assays, primarily due to their highsensitivity and non-radioactivity. The hydrolytic enzymes includephosphatases, glycosidases, peptidases, proteases, and esterases. Byfar, most commonly used are phosphatases and glycosidases. For instance,alkaline phosphatase has been extensively used as a label in variousenzyme-linked immunosorbent assays (ELISAs) due to its high turn-overrate, excellent thermal stability and ease of use. Many glycosidases,such as β-galactosidase and β-glucuronidase, have also been used inELISA due to their very high selectivity for the hydrolysis of theirpreferred substrates. On the other hand, some hydrolytic enzymes playimportant functions by themselves in biological processes of the humanbody and microorganisms. Therefore, direct detection of these markers isanother important aspect of diagnostics.

In connection with the detection of hydrolytic enzymes, there are threetypes of substrates: chromogenic, fluorogenic and chemiluminescentsubstrates. Among them, chemiluminescent substrates offer the bestenzyme detection sensitivity due to the intrinsic advantages of higherdetectability of chemiluminescent product, or lower substrate andinstrumental backgrounds, and less interference from biological samples.Therefore, there has been a steady trend towards developingchemiluminescent substrates and applying them in a variety ofdiagnostics.

Stable Dioxetanes

One class of widely used chemiluminescent substrates for hydrolyticenzymes are stable dioxetanes (Bronstein et al., U.S. Pat. Nos.4,931,223, 5,112,960, 5,145,772 and 5,326,882; Schaap et al., U.S. Pat.Nos. 5,892,064, 4,959,182 and 5,004,565; and Akhavan-Tafti et al., U.S.Pat. No. 5,721,370). Here, the thermally stable protective group on thephenolic moiety of the dioxetane substrates is cleaved by a hydrolyticenzyme of interest, such as alkaline phosphatase (AP) orβ-galactosidase, depending on whether the protective group is aphosphoryl or β-D-galactopyranosidyl group. The newly generateddioxetane phenoxide anion undergoes auto-decomposition to amethoxycarbonylphenoxide in an electronically excited state. The latterthen emits light at λmax˜470 nm.

In an aqueous environment where virtually all biological assays areperformed, the decomposition of the dioxetanes produceschemiluminescence in a very low quantum yield, typically about 0.01%,and a slow kinetics with t_(1/2) 1˜10 minutes. This is quite differentfrom the decomposition of the dioxetanes in an organic environment. Forinstance, the dioxetane having the phenol moiety protected by an acetylgroup or a silyl group, upon treatment with a base or fluoride, exhibitsquantum yield up to 25% in DMSO and 9.4% in acetonitrile, respectively,and t_(1/2) is about 5 sec. at 25° C. Schaap, et al., TetrahedronLetters, 28(11), 1155, (1987) and WO 90/07511 A.

Voyta et al., (U.S. Pat. No. 5,145,772) disclosed a method ofintermolecular enhancement of quantum yield of the dioxetane productsusing polymeric ammonium salts, which provide a hydrophobic environmentfor the phenoxide produced by the enzyme.

Akhavan-Tafti et al., reported methods of intermolecular enhancement ofquantum yield of the dioxetane products using polymeric phosphoniumsalts (U.S. Pat. No. 5,393,469) and dicationic surfactants (U.S. Pat.Nos. 5,451,347 and 5,484,556).

Schaap et al., (U.S. Pat. Nos. 4,959,182 and 5,004,565) disclosedanother method for increasing quantum yield of the dioxetane productsusing fluorescent co-surfactants as energy acceptors. The resonanceenergy embodied in the excited phenoxide produced by the enzyme iseffectively transferred to the fluorescent co-surfactants. Instead ofemitting light at λmax 470 nm characteristic of the dioxetane, thissystem emits light at λmax 530 nm as a result of energy transfer to thehighly efficient fluorophore, fluorescein.

Another approach for improving quantum yield of the dioxetanes,disclosed by Schaap et al., (U.S. Pat. No. 5,013,827), is to covalentlyattach a fluorophore having high quantum yield to the light emittingphenoxide moiety. The resonance energy from the excited phenoxide isintramolecularly transferred to the attached fluorophore. The latter inturn emits light at its own wavelength. It is claimed that suchdioxetane-fluorophore conjugates exhibit quantum yield as high as 2%.

Wang et al., (WO 94/10258) unveiled a class of electron-rich,aryl-substituted dioxetane compounds in which the aryl group ispoly-substituted with a suitable electron-donating group so that intenseluminescence is observed.

Akhavan-Tafti et al., (U.S. Pat. No. 5,721,370) provided a group ofstable chemiluminescent dioxetane compounds with improved watersolubility and storage stability. The compounds are substituted with twoor more hydrophilic groups disposed on the dioxetane structure and anadditional fluorine atom or lower alkyl group.

Schaap et al., (U.S. Pat. No. 5,892,064) disclosed a class ofchemiluminescent dioxetane compounds substituted on the dioxetane ringwith two nonspirofused alkyl groups.

Urdea at al. (EP Application 0401001 A2) described another sub-class ofdioxetane compounds that can be triggered by sequential treatment withtwo different activating enzymes to generate light. The system rests onthe principle that the dioxetane substrates have two protecting groupsthat can be removed sequentially by different processes to produce anexcited phenoxide, and the removal of the first protecting group istriggered by the enzyme used as a label in the assay.

Luminol Substrates

Sasamoto at al., Chem. Pharm. Bull., 38(5), 1323 (1990) and Chem. Pharm.Bull., 39(2), 411 (1991), reported thato-aminophthalhydrazide-N-acetyl-β-D-glucosaminide (Luminol-NAG) and4′-(6′-diethylaminobenzofuranyl)-phthalhydrazide-N-acetyl-β-D-glucosaminide,both being the non-luminescent forms of luminol, are substrates ofN-acetyl-β-D-glucosaminidase. Upon the action of the enzyme on thesesubstrates, luminol or luminol derivative is generated, which then canbe detected by triggering with 0.1% hydrogen peroxide and a peroxidase(POD) or Fe(III)-TCPP complex catalyst to release a chemiluminescentsignal.

Enzyme-Modulated Protected Enhancer and Anti-Enhancer

Kricka (U.S. Pat. No. 5,306,621) disclosed that light intensity ofcertain peroxidase-catalyzed chemiluminescent reactions can be modulatedby AP that acts on a pro-enhancer or a pro-anti-enhancer. For example,the intensity of a chemiluminescent reaction containing luminol,horseradish peroxide and hydrogen peroxide can be enhanced by anenhancer (4-iodophenol) liberated by the enzymatic action of AP on apro-enhancer (4-iodophenol phosphate), thus enabling AP to be assayed.Alternatively, in the same above reaction where additional enhancer(4-iodophenol) is present, the light intensity can be decreased by ananti-enhancer (4-nitrophenol) generated by the enzymatic action of AP ona pro-anti-enhancer (4-nitrophenol phosphate). In the latter format, thepresence of AP can be assayed by measuring the reduction in the lightintensity.

Similar to the above, an assay using enzyme-triggerable protectedenhancer for quantitation of hydrolytic enzymes was also unveiled byAkhanvan-Tafti in EP Application 0516948 A1.

Akhanvan-Tafti et al., (WO 96/07911) disclosed another method ofdetecting hydrolytic enzymes based on the principle of the protectedenhancer, where the light emitting species is generated from theoxidation of acridan by peroxidase and peroxide.

Acridene Enol Phosphate

Akhanvan-Tafti et al., (U.S. Pat. No. 5,772,926 and WO 97/26245)disclosed a class of heterocyclic, enol phosphate compounds representedby the non-luminescent acridene enol phosphate. Upon the reaction with aphosphatase enzyme, acridene enol phosphate is converted to thedephosphoryl enolate, which reacts with molecular oxygen to producelight (see scheme below). It was also disclosed that the light outputwas greatly enhanced by the addition of a cationic aromatic compound tothe assay system.

Other Chemiluminescent Substrates for Hydrolytic Enzymes

Vijay (U.S. Pat. No. 5,589,328) disclosed chemiluminescent assays thatdetect or quantify hydrolytic enzymes, such as alkaline phosphatase,that catalyze the hydrolysis of indoxyl esters. The assay includes thesteps of reacting a test sample with an indoxyl ester and thenimmediately or within a short time (typically less than about 15minutes) measuring the resulting chemiluminescence. The resultingchemiluminescence may be amplified by adding a chemiluminescentenhancing reagent.

Among the above chemiluminescent hydrolytic enzyme methods, thedioxetane system appears to be the most sensitive detection system andtherefore has been increasingly used in various assays. Despite itswidespread use, this system has an inherent drawback in that backgroundchemiluminescence in the absence of enzyme is observed due to slowthermal decomposition and non-enzymatic hydrolysis of the dioxetane.Another intrinsic disadvantage is that the phenoxide, once generated byenzymatic reaction, is extremely unstable and readily undergoesdecomposition to release light. In this sense, the phenoxide, the lightemitting species, is never “accumulated” during the enzymatic reaction.Therefore, it is one of the major goals of this invention to provide newchemiluminescent substrates whose derived chemiluminescent products havedistinguishable emission profiles and whose total signal can beaccumulated during the enzymatic reaction, thereby providing analternative and sensitive detection method for hydrolytic enzymes.

BRIEF SUMMARY OF THE INVENTION

It is an object of this invention to provide chemiluminescent compoundsthat are substrates of hydrolytic enzymes. Said chemiluminescentsubstrates upon treatment with a hydrolytic enzyme convert to thecorresponding chemiluminescent products that have distinctly differentlight emission characteristics.

It is another object of this invention to provide acridinium-basedchemiluminescent compounds that are substrates of hydrolytic enzymes.Said chemiluminescent substrates contain a phenolic moiety or enolmoiety in the molecule that is masked by a group, which is thermally andhydrolytically stable. Upon treatment with a hydrolytic enzyme, saidchemiluminescent substrates convert to the correspondingacridinium-based chemiluminescent products that have distinctlydifferent light emission characteristics.

It is also an object of this invention that said chemiluminescentsubstrates and products have distinctly different light emissioncharacteristics, thereby allowing the separation or distinction of thesignal of the substrate from the signal of the product, or vice versawhen both substrate and product are present in the same test vessel.

It is another object of this invention that said chemiluminescentproducts generated by hydrolytic enzymes have emission maxima differentfrom those of their corresponding chemiluminescent substrates.

It is another object of this invention that said chemiluminescentproducts generated by hydrolytic enzymes have quantum yields differentfrom those of their corresponding chemiluminescent substrates.

It is another object of this invention that said chemiluminescentproducts generated by hydrolytic enzymes have light-emitting kineticsdifferent from those of their corresponding chemiluminescent substrates.

It is another object of this invention that said chemiluminescentproducts generated by hydrolytic enzymes have physical and chemicalproperties different from those of their corresponding chemiluminescentsubstrates. Said physical and chemical properties include, but are notlimited to, the fundamental net charge distribution, dipole moment,π-bond orders, free energy, or the apparenthydrophobicity/hydrophilicity, solubility, affinity and other propertiesthat are otherwise apparent to those who are skilled in the art.

It is another object of this invention that said chemiluminescentsubstrates are structurally manipulated such that the distinction of thelight emission characteristics can be further enlarged.

It is yet another object of this invention that chemiluminescentproducts resulting from the action of hydrolytic enzymes onchemiluminescent substrates do not undergo substantial decompositionduring the enzymatic reaction, and thus can be accumulated untiltriggered by a light-releasing reagent.

The combined objects and advantages of this invention indicated aboveare attained by:

A. Firstly, a chemiluminescent substrate of hydrolytic enzyme having thefollowing general Formula I, as follows:Lumi-M-P  Formula Iwhere “Lumi” is a chemiluminescent moiety capable of producing light (a)by itself, (b) with MP attached and (c) with M attached. Lumi includes,but is not limited to, chemiluminescent acridinium compounds (e.g.,acridinium esters, acridinium carboxyamides, acridinium thioesters andacridinium oxime esters), benzacridinium compounds, quinoliniumcompounds, isoquinolinium compounds, phenanthridinium compounds, andlucigenin compounds, or the reduced (e.g., acridans) or non-N-alkylatedforms (e.g., acridines) of the above, as well as spiroacridan compounds,luminol compounds and isoluminol compounds and the like. M is amultivalent heteroatom having at least one lone pair of electronsselected from oxygen, nitrogen and sulfur, directly attached to thelight emitting moiety of Lumi at one end and to P at the other end.(When M alone is attached to Lumi to form Lumi-M, it does, of course,have either a proton or a counterion associated with it or is in theform of an ion.) P is a group that can be readily removed by hydrolyticenzymes, as discussed in more detail hereinafter.

The light emitting moiety of Lumi is well known. For example, when Lumiis an acridinium compound or luminol, the light emitting moiety is theacridinium nucleus or phthaloyl moiety, respectively.

B. Secondly, an enzymatic reaction having the following general reactionA, as follows:

where HE is a hydrolytic enzyme, such as phosphatase, glycosidase,peptidase, protease, esterase, sulfatase or guanidinobenzoatase.Lumi-M-P is a chemiluminescent substrate of a hydrolytic enzyme. Lumi-Mis a chemiluminescent product having physical and/or chemical propertiesdifferent from those of Lumi-M-P. Said physical and/or chemicalproperties include emission wavelength, quantum yield, light emissionkinetics, fundamental net charge distribution, dipole moment, π-bondorders, free energy, or apparent hydrophobicity/hydrophilicity,solubility, affinity and other properties.

C. Light releasing reactions which take place on both Lumi-M-P andLumi-M.

It is one object of this invention to provide novel light-releasingreagent compositions and reagent addition protocols for triggering lightemission from chemiluminescent substrates and products that surprisinglyresult in better distinction between the signals of the chemiluminescentsubstrates and products. Said light-releasing reagent compositions canbe a single reagent and/or multiple reagents, and addition of saidmultiple reagents to the reaction vessel can be synchronous orsequential. According to the invention, the light releasing reactionsmay take place on both Lumi-M-P and Lumi-M, as shown in Formula I.

It is another object of this invention that said light-releasing reagentcompositions comprise one or more peroxides or peroxide equivalents, andsaid peroxides or peroxide equivalents include, but are not limited to,hydrogen peroxide.

It is yet another object of this invention that said light-releasingreagent compositions interact with said chemiluminescent substrate andproduct differentially so that the differentiation between the twosignals is optimized.

It is yet another object of this invention that said light-releasingreagent compositions contain one or more enhancers selected fromorganic, inorganic or polymeric compounds with a broad range ofmolecular weights, which differentially enhance the light output fromeither the substrate or the product.

It is yet another object of this invention that said light-releasingreagent compositions contain also one or more quenchers, blockers orinhibitors selected from organic, inorganic or polymeric compounds witha broad range of molecular weights such that they differentially quench,block or reduce the light output from either the substrate or theproduct.

The combined objects and advantages of this invention indicated aboveare attained by a light-releasing reagent composition. Said lightreleasing reagent composition consists of two separate reagents, whichare sequentially added to a solution containing the chemiluminescentsubstrate and/or product. The first reagent contains acidic hydrogenperoxide solution, and the second reagent contains an alkaline solutionwith one or more detergents. Alternatively, to the advantage of betterdistinction between the signals of certain chemiluminescent substratesand their products, the first reagent contains an alkaline solution withone or more detergents, and the second reagent contains hydrogenperoxide solution.

It is another object of this invention to provide optimal lightdetection methods for said chemiluminescent reactions, so that thedifferentiation between light emissions of the substrate and product isoptimized for specific applications. Said optimal detection methodscomprise the use of a light detection apparatus, which includes aluminometer, a Charge-Coupled Device (CCD) camera, an X-ray film, and ahigh-speed photographic film. Said luminometer comprises ablue-sensitive photomultiplier tube (PMT), or a red-sensitive PMT, orother PMTs optimized for specific applications. Said optimal lightdetection methods also include the use of an optical filtering device toblock or reduce unwanted light emission either from the substrate orfrom the product. Said optimal detection methods further include amethod and/or a device for detecting or registering light in asequential manner that eliminates or reduces unwanted signal from thesubstrate.

The combined objects and advantages of this invention indicated aboveare attained by one or more light detection methods. One preferred lightdetection method comprises the use of a PMT and a long wave pass filterin an enzymatic reaction to detect the chemiluminescent signal from theproduct that emits light at a wavelength longer than that of thesubstrate. Another said light detection method comprises the use of ared-sensitive PMT at low temperature (i.e., below 4° C.) and a long wavepass filter to improve the detectability of the chemiluminescent productthat emits light at a wavelength longer than that of the substrate.Still another said light detection method comprises the use ofblue-sensitive PMT and a short wave pass filter to detect the decreaseof chemiluminescent signal from the substrate whose emission wavelengthis shorter than that of the product. Yet another said light detectionmethod comprises the use of a PMT with or without an optical filter,within a fixed light measuring time, to detect the decrease ofchemiluminescent signal from the substrate whose emission kinetics isfaster than that of the product.

It is yet another object of this invention to provide methods and assayscomprising the use of one or more said chemiluminescent substrates fromFormula I, one or more said hydrolytic enzymes, one or more saidlight-releasing reagent compositions, and one or more said optimal lightdetection methods.

It is yet another object of this invention to provide methods and assaysin using one or more said chemiluminescent substrates to detect,qualitatively or quantitatively, the presence of one or more saidhydrolytic enzymes or enzyme conjugates that are present either aslabels or as markers of biological samples.

It is a further object of this invention to provide a method thatutilize one or more said chemiluminescent substrates from Formula I, andone or more said labeled hydrolytic enzymes to detect, qualitativelyand/or quantitatively, the presence of one or more analytes.

Finally, it is also an object of this invention to provide syntheticmethods and intermediates related to the syntheses of saidchemiluminescent substrates.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

FIGS. 1A-1F show structures of chemiluminescent substrates(2-phos-acridinium esters), capable of short wavelength emission, andtheir corresponding chemiluminescent products (2-hydroxyl-acridiniumesters), capable of long wavelength emission.

FIGS. 1G-1L show structures of chemiluminescent substrates(2-phos-acridnium esters) capable of short wavelength emission, andtheir corresponding chemiluminescent products (2-hydroxyl-acridiniumesters), capable of long wavelength emission, including spiro compounds(FIGS. 1K-1L).

FIGS. 1M-1P show structures of chemiluminescent substrates capable oflong wavelength emission and its corresponding products capable of shortwavelength emission.

FIGS. 1Q-1R show structures of chemiluminescent substrates and itscorresponding product, which have different emission kinetics.

FIGS. 2A-2P are the emission spectra of chemiluminescent substrates(2-phos-acridinium esters) and their corresponding chemiluminescentproducts (2-hydroxyl-acridinium esters), as well as spectra ofchemiluminescent substrates capable of long emission and theircorresponding products capable of short emission, determined by a FastScanning Spectral System (FSSS) The emission spectra of chemiluminescentsubstrates and the corresponding products, which have different lightemission kinetics, are also shown.

FIG. 3 is a plot of wavelength vs. quantum yield of Hamamatsuphotomultiplier tube R268.

FIG. 4 is a plot of wavelength vs. quantum yield of Hamamatsuphotomultiplier tube R2228P.

FIG. 5 is a plot of wavelength vs. quantum yield of the thinned,back-illuminated Charge Couple Device (thinned CCD).

FIG. 6 is a profile of the transmittance of Corion cut-on long wave passfilter LL650 (Lot No. CFS-002645).

FIG. 7 is a profile of the transmittance of Corion long wave pass filterLL700.

FIGS. 8 and 9 are plots of stabilities of chemiluminescent product(2-OH-DMAE) in pH 10.5, 100 mM Tris buffer containing 1 mM MgCl₂ as afunction of time.

FIG. 10 is a plot showing the detectability of alkaline phosphataseusing 2-Phos-DMAE as the substrate. The enzyme reaction was incubated at45° C. for 0.5 hour. The reaction mixture was flashed with 0.25 N NaOHcontaining 0.5% CTAB immediately followed by 0.5% H₂O₂. The light outputwas measured for 2 seconds on MLA-1 equipped with R268 PMT and two LL650long wave pass filters.

FIG. 11 is a plot showing the detectability of alkaline phosphataseunder the same reaction condition as described in FIG. 10, with theexception that the light output was measured on MLA-1 equipped withR2228P PMT and two LL650 long wave pass filters, and the unit waspre-cooled to 4° C.

FIG. 12 is a plot showing the detectability of alkaline phosphataseusing 2-Phos-DMAE as the substrate. The enzyme reaction was incubated at45° C. for 1 hour. The reaction mixture was flashed with 0.25 N NaOHcontaining 0.5% CTAB immediately followed by 0.5% H₂O₂. The light outputwas measured for 2 seconds on MLA-1 equipped with R2228P PMT and a LL700long wave pass filters, and the unit was pre-cooled to 4° C.

FIG. 13 is a plot of showing the detectability of alkaline phosphataseusing 4′-Phos-AE (FIG. 1Q) as the substrate.

FIG. 14 is a plot of the standard curve of TSH immunoassay using2-phos-DMAE (1) as the chemiluminescent substrate and alkalinephosphatase as the label.

DETAILED DESCRIPTION OF THE INVENTION

It has been discovered that certain novel chemiluminescent compoundshaving a general Formula I are substrates of hydrolytic enzymes, withthe structure of Formula I shown as follows:Lumi-M-P  Formula I

As shown in Formula I, Lumi is defined as a chemiluminescent moietycapable of producing light (a) by itself, (b) with MP attached and (c)with only M attached. M is defined as a multivalent heteroatom having atleast one lone pair of electrons selected from oxygen, nitrogen andsulfur, wherein M is directly attached to the light emitting moiety ofLumi at one end and to P at the other end. P is a group that can bereadily removed by hydrolytic enzymes. When M alone is attached to Lumito form Lumi-M, M has either a proton or a counterion associated with itor is in the form of an ion.

The chemiluminescent moiety of Lumi includes, but is not limited to,acridinium compounds (including acridinium esters (Law et al., U.S. Pat.No. 4,745,181), acridinium carboxyamides (Mattingly et al., U.S. Pat.No. 5,468,646 and Kinkel et al., J. Biolumin. Chemilumin., 4, 136-139,(1989)), acridinium thioesters (Kinkel et al., J. Biolumin. Chemilumin.,4, 136-139, (1989)) and acridinium oxime esters (Ghitti et al., WO98/56765)) benzacridinium compounds, quinolinium compounds,isoquinolinium compounds, phenanthridinium compounds, and lucigenincompounds, or the reduced (e.g., acridans) or non-N-alkylated (e.g.,acridines) forms of the above, spiroacridan compounds (Singh et al., WO94/02486), luminol compounds, and isoluminol compounds, as shown below.

Hydrolytic enzymes that may be used in Reaction A, shown previously,include, but are not limited to, phosphatases: alkaline phosphatase,acidic phosphatase, phospholipase, phosphodiesterase, andpyrophosphatase; glycosidases: β-galactosidase, α-galactosidase,α-(D-)-glucosidase, β-glucosidase, β-glucuronidase, α-manno-sidase,N-acetyl-β-D-glucosaminidase, neuraminidase, cellulase, andβ-fucosidase; peptidases and proteases: dipeptidylpeptidases I, II andIV, plasminogen activator, calpain, elastase, trypsin, cathepsins B, C,L and O, urokinase, granzyme A, prostatin, thrombin, trypase, follipsin,kallikrein, plasmin, prohormone thiol protease, amyloid A4-generatingenzymes, human adenovirus proteinase, kallikrein, and HIV protease;esterases: cholinesterase, and lipase; and sulfatase, andguanidinobenzoatase.

Being thermally and hydrolytically stable in an aqueous environment, theinventive chemiluminescent substrates undergo readily hydrolyticreaction in the presence of hydrolytic enzymes, and the resultingproducts, as represented by Lumi-M in Reaction A, are alsochemiluminescent. It has also been unexpectedly discovered that thelight emission characteristics (emission wavelength, kinetics, andquantum yield) of the products differ significantly from those of theircorresponding chemiluminescent substrates due to the change in some ofthe chemical and physical properties of the chemiluminescent productsgenerated by hydrolytic reaction. Those chemical and physical propertiesinclude the fundamental net charge distribution, dipole moment, π-bondorders, free energy, or the apparent hydrophobicity/hydrophilicity,solubility, and affinity, etc. As a result, a discernible signal due toone or more of these differences is generated, and thus is useful forqualitative or quantitative determination of the specific hydrolyticenzyme that is involved in the Reaction A, shown previously.

1. Substrates and Products Having Different Emission Wavelength

One preferred class of chemiluminescent substrates selected from FormulaI in this invention is related to novel chemiluminescent acridiniumcompounds represented in Formula II, shown below, with said compoundscapable of being chemically triggered to produce light:

As shown in Formula II, P is preferably a group that is thermally andhydrolytically stable in aqueous medium, but is readily removable by ahydrolytic enzyme to form Lumi-M; and M is preferably a multivalentheteroatom having at least one lone pair electrons selected from oxygen,nitrogen and sulfur, and a strong ability of donating electrons to theacridinium nucleus after the removal of P. Preferably, after the removalof the protective group (P) by a hydrolytic enzyme, M becomes ionizablein the medium of the reaction to bear a negative charge, thus stronglydonating electrons to the acridinium ring system.

As shown in Formula II, R₁ is preferably an alkyl, alkenyl, alkynyl oraralkyl group containing from 0 to 20 heteroatoms; more preferably R₁ ismethyl and most preferably R₁ is sulfoalkyl or an alkyl containing-oneor more hydrophilic groups selected from sulfonate, sulfate, —CO₂H,phosphonate, ethylene glycol, polyethylene glycol, quaternary ammonium(—N⁺R₃), or any groups containing one or more of the above (—N⁺R₃). Theinclusion of the hydrophilic moiety in R₁ serves to increase watersolubility of the molecule.

As shown in Formula II, C₁, C₃, C₆, and C₈ peri-positions of theacridinium nucleus may be unsubstituted or substituted. When one or moreof said positions are substituted, the substituents (R_(2a), R_(2b),R_(3b), and R_(3d), respectively) are identical or different, selectedfrom —R, substituted or unsubstituted aryl (ArR or Ar), halides, nitro,sulfonate, sulfate, phosphonate, —CO₂H, —C(O)OR, cyano (—CN), —SCN, —OR,—SR, —SSR, —C(O)R, —C(O)NHR, ethylene glycol, or polyethyelene glycol. Ris selected from the group consisting of alkyl, alkenyl, alkynyl aryl,and aralkyl containing from 0 to 20 heteroatoms.

As shown in Formula II, the C₄, C₅, and C₇ peri-positions of theacridinium nucleus may be unsubstituted or substituted. When one or moreof said positions are substituted, the substituents (R_(2c), R_(3a), andR_(3c), respectively) are identical or different and are defined thesame as R_(2a), R_(2b), R_(3b), and R_(3d). Alternatively, one ofR_(2c), R_(3a) and R_(3c) may be defined as M-P. In this case, C₂peri-position may be unsubstituted or substituted. When it issubstituted, the substituents may be defined as R_(2a), R_(2b), R_(3b),and R_(3d).

Alternatively, as shown in Formula II, any two adjacent substituents atthe acridinium nucleus peri-positions can be linked as in the followingexamples, so as to form additional unsaturated carbocyclic and/orheterocyclic rings fused to the attached acridinium nucleus, as shownbelow.

As provided in Formula II, A⁻ is a counter ion for the electroneutralityof the quaternary nitrogen of the acridinium compounds, which isintroduced as a result of quarternerization of the intermediateacridines with alkylating agents, or due to anionic exchange whichoccurs during the subsequent synthetic steps or in the following work-upof reaction mixtures and purification of the desired compounds in aliquid phase containing excess amounts of other anions. Examples of suchcounter ions include CH₃SO₄ ⁻, FSO₃ ⁻, CF₃SO₃ ⁻, C₄F₉SO₃ ⁻, CH₃C₆H₄SO₃⁻, halide, CF₃COO⁻, CH₃COO⁻, and NO₃ ⁻. A⁻ is typically not present ifthe R₁ substituent contains a strongly ionizable group that can form ananion and pair with the quaternary ammonium cationic moiety.

X is nitrogen, oxygen or sulfur.

When X is oxygen or sulfur, Z is omitted and Y is a substituted orunsubstituted aryl group, and preferably Y is a polysubstituted arylgroup of the formula III:

As shown in Formula III, R₄ and R₈ may be identical or different andidentified as an alkyl, alkenyl, alkynyl, alkoxyl (—OR), alkylthiol(—SR), or substituted amino groups that serve to stabilize the —COX—linkage between the acridinium nucleus and the Y moiety, through stericand/or electronic effect; preferably R₄ and R₈ are short chain alkylgroups having from 1 to 10 carbon atoms, more preferably a methyl group,or at least one of R₄ and R₈ is as defined while the other is a hydrogenor an atom selected from halides. R₅, R₆ and R₇ may be identical ordifferent, selected from hydrogen, —R, substituted or unsubstitutedaryl, halides, amino, —NHR, NR₂, quaternary ammonium (—N⁺R₃), hydroxyl,nitro, nitroso, sulfonate, sulfate, cyano (—CN), phosphonate, CO₂H,—SCN, —OR, —SR, —SSR, —C(O)R, —C(O)NHR, —NHC(O)R, ethylene glycol, orpolyethyelene glycol. Preferably, R₅, R₆ and R₇, identical or different,are a hydrophilic group selected from sulfonate, sulfate, —CO₂H,phosphonate, ethylene glycol, polyethylene glycol, quaternary ammonium(—N⁺R₃), or any groups containing one or more of the above hydrophilicmoiety, which serves to increase water solubility of the molecule.

Any adjacent two groups of R₄ to R₈ in Formula III can form one or moreadditional fused hydrocarbon aromatic rings or heteroaromatic rings withor without substitutions. The additional fused hydrocarbon aromaticrings and heteroaromatic rings include, but are not limited to, benzene,naphthlene, pyridine, thiophene, furan, and pyrrole, etc.

Alternatively, Formula II can represent another class of Lumi-M-P whereLumi is an acridinium oxime ester (as described in WO 98/56765 herebyincorporated by reference), and M-P are as defined above. When the Lumiis an acridinium oxime ester, said X, Y and Z of Formula II areseparately defined.

As shown in Formula II, when X is oxygen, Z is omitted and Y is definedabove or —N═CR₉R₁₀, wherein R₉ and R₁₀, are identical or different, andare selected from hydrogen, substituted or non-substituted aryl, alkyl,alkenyl, alkynyl, halide, alkoxyl and aryloxy groups.

As shown in Formula II, when X is nitrogen, then Z is —SO₂—Y′, Y′ hasthe same definition of Y as defined above, and Y and Y′ may be the sameor different. Additionally, Y itself can be a branched or straight-chainalkyl containing from 1 to 20 carbons, halogenated or unhalogenated, orcan be a substituted aryl, or heterocyclic ring system.

Thus, given the alternatives discussed above, X, Y and Z in Formula IIcan be defined as follows:

-   -   X is nitrogen, oxygen or sulfur; such that,    -   when X is oxygen, Z is omitted and Y is a substituted or        unsubstituted aryl group or —N═CR₉R₁₀, wherein R₉ and R₁₀ may be        the same or different and are selected from hydrogen,        substituted or non-substituted aryl, alkyl, alkenyl, alkynyl,        halide, alkoxyl and aryloxy groups;    -   when X is sulfur, Z is omitted and Y is a substituted or        unsubstituted aryl group;    -   when X is nitrogen, Z is —SO₂—Y′, Y′ being defined the same as Y        above; Y is as defined above or can be a branched or        straight-chain alkyl containing 0 to 20 carbons, halogenated or        unhalogenated, or a substituted aryl, or heterocyclic ring        system; and Y and Y′ can be the same or different.

Structurally closely related to chemiluminescent acridinium compounds ofFormula II are their reduced forms, chemiluminescent acridan compounds,represented as Formula IV, where all substitutions are as defined inFormula II. These compounds can be chemically triggered to produce lightvia formation of acridinium intermediates and/or via direct oxidation toform electronically excited products, which are the same products ofacridinium compounds of Formula II.

Another class of chemiluminescent substrates structurally related toacridinium substrates of Formula II are spiroacridan compounds havingFormula V, wherein X₁ and X₂ may be identical or different, are selectedfrom the group consisting of oxygen, sulfur and nitrogen, and when X₁and/or X₂ are oxygen or sulfur, Z₁ and/or Z₂ are omitted, when X₁ and/orX₂ are nitrogen, Z₁ and/or Z₂ are hydrogen, alkyl, aryl or —SO₂—Y′; G isa group connecting X₁ and X₂ to form a ring having 5 to 10 members; andR₁, R_(2a-c), R_(3a-d), M and P are defined in Formula II.

Within the chemiluminescent spiroacridan substrates of Formula V is asubclass shown as Formula VI below, where G is singly or multiplysubstituted (R₁₁) or unsubstituted aromatic ring with 0-3 heteroatoms,wherein R₁₁ is the group selected from hydrogen, —R, substituted orunsubstituted aryl (ArR or Ar), halides, nitro, sulfonate, sulfate,phosphonate, —CO₂H, —C(O)OR, cyano (—CN), —SCN, —OR, —SR, —SSR, —C(O)R,—C(O)NHR, ethylene glycol, or polyethyelene glycol, and said subclass ofsubstrates having Formula VI:

Preferably, novel chemiluminescent acridinium substrates of the presentinvention of Formula II are a subclass of compounds represented byFormula VII below, wherein multivalent heteroatom M is oxygen (O), andgroup P is a phosphoryl group, —PO₃Na₂, where two sodium cations, whosepurpose is solely for the eletroneutrality of the molecule, can beexchanged independently with hydrogen, potassium, magnesium, calcium, orother cationic

The preferred hydrolytic enzyme (HE) as shown in Scheme I is ahydrolytic phosphatase. Particularly preferred is the use of hydrolyticphosphatase with a compound defined by Formula VII.

Structurally closely related to preferred chemiluminescent acridiniumcompounds of Formula VII are their reduced forms, chemiluminescentacridans, represented as Formula VIII, where all substitutions are asdefined in Formula VII. The compounds of Formula VII may be chemicallytriggered to produce light via formation of acridinium intermediatesand/or via direct oxidation to form electronically excited products,which are the same products of acridinium compounds of Formula VII.

Another subclass of the compounds structurally related to preferredchemiluminescent acridinium compounds of Formula VII are spiroacridancompounds of Formula IX, wherein, X₁, X₂, Z₁, Z₂, R′, R₁, R_(2a-c),R_(3a-d), M and P are as defined in Formula VI, and two sodium cationsare as defined in Formula VII.

One of the preferred compounds selected from Formula VII, in whichR_(2a-c), R_(3a-d), are hydrogen, R₁ is methyl, X is oxygen, Z isomitted, and Y is a polysubstituted aryl moiety of Formula III where R₄and R₈ are methyl, R₅ and R₇ are hydrogen, and R₆ is carboxyl (—CO₂H),is 2-Phos-DMAE, whose structure is shown as 1. (See FIG. 1A. Note:Structure 1 below is also shown in FIG. 1A, attached. Similarly, otherstructures in this application are referred to by numbers whichcorrespond to the appropriate sequence number in FIG. 1.)

As disclosed in the section of Examples, 2-Phos-DMAE (1) is an excellentsubstrate of hydrolytic alkaline phosphatase (AP). In alkaline aqueousmedium of a wide pH range, it is readily dephosphorylated by AP to form2-OH-DMAE (2) as illustrated in Reaction B. Both 1 and 2 arechemiluminescent. As disclosed in FIGS. 2A-2B and also in Table 1, ithas been unexpectedly discovered that 1 and 2 emit light at differentemission maxima when they are treated with hydrogen peroxide in strongalkaline solution, respectively. Specifically, compound 1 emits astrong, visible blue light at λmax 478 nm while compound 2 emits astrong, visible orange light at λmax 602 nm, thus resulting in abathochromic shift of emission maximum by 128 nm. Several pairs ofanalogs (3 and 4, 5 and 6, 7 and 8), as well as a pair of spiroacridans(11 and 12), whose structures are given in FIG. 1, also have the samelight emission characteristics. Compounds 7 and 8 also carry additionalhydrophilic groups on the nitrogen of the acridinium nucleus.

As illustrated in Reaction C, the chemiluminescent reaction of theacridinium compound is triggered by hydrogen peroxide in strong alkalinesolution. Formation of the high energy dioxetanone follows the departureof the leaving group LG. The highly strained dioxetanone intermediatedecomposes to the acridone, a portion of which is formed in anelectronically excited state. When the excited acridone reverts to aground state, light emission occurs. The emission wavelength isdetermined by the energy gap between the first excited state and theground state, which in turn is determined by the specific structure ofthe acridone having various functional groups T. As disclosed in WO00/09487, which is fully incorporated herein by reference, there areseveral factors which can influence the energy gap between the firstexcited state and the ground state of the acridone, and thus affect theemission wavelength. One of the factors is the direct attachment of therequisite functional group T to one of the peri-positions of theacridinium nucleus. It has been discovered and disclosed in theaforementioned WO 00/09487 that when a hydroxyl group is placed at the(2) or (7) position, such as in 2-OH-DMAE (2), the compound emits lightat λmax 604 nm in strong alkaline solution. This is because in a strongalkaline solution, the hydroxyl group becomes deprotonated, and theresulting negatively-charged oxy anion exerts a strong electron donatingeffect to the acridinium nucleus, consequently decreasing the energy gapbetween the first excited state and the ground state of the acridone,which causes a bathochromic shift of light emission to 602 nm from 430nm of the unsubstituted acridinium compound.

In this invention, a phosphoryloxy group at the (2) position of 1 servesas a masked hydroxyl group. This phosphoryloxy group is thermally andhydrolytically stable in aqueous medium with a wide range of pHs wherevirtually all hydrolytic enzymatic reactions take place. It is alsostable in a mixture of hydrogen peroxide and strong alkaline solutionfor a length of time that is needed for converting acridinium compoundsto the light emitting acridones. Because the phosphoryloxy group in 1 isvirtually intact in the absence of hydrolytic enzyme during the reactionand also stable to hydrogen peroxide and strong alkaline solution, theelectron-donating effect of the directly attached oxygen at the (2)position towards the acridinium nucleus is significantly diminished.Thus, no bathochromic shift of emission wavelength occurs. This is insharp contrast to the situation of 2-OH-DMAE (2), the product of thereaction in the presence of hydrolytic enzyme. In the latter case, thehydroxyl group at the (2) position is ionized to a negatively-chargedoxy anion, which has a strong electron donating effect to the acridiniumnucleus, thus causing a significant bathochromic shift of emissionwavelength.

It would be desirable that in a system using 1 as a chemiluminescentsubstrate, a specific hydrolytic enzyme such as AP that is either usedas a label or present as a marker of a biomolecule converts a shortwavelength emitting 1 to a long wavelength emitting 2. Indeed, upontreatment with hydrogen peroxide and strong alkaline solution, thereaction mixture containing both 1 and 2 produces a mixture of lightsignals each with its own maximum at 478 nm and 602 nm, respectively.The decrease in the short signal (λmax 478 nm) or the increase in thelong signal (λmax 602 nm) directly correlates to the amount of theenzyme.

Another preferred compound selected from Formula VII is2-Phos-7-MeO-DMAE (9). Unexpectedly, the dephos-phorylated product,2-OH-7-MeO-DMAE (10) in Reaction D was found to have 60-70 nm longeremission maxima than the unmethoxylated compound 2 (2-OH-DMAE), whileboth of the corresponding 2-phosporylated forms have nearly the samelight emission maxima. Thus, the 2-Phos-7-MeO-DMAE and 2-OH-7-MeO-DMAEpair of chemiluminescent substrate and product provide effectivelyfurther improved distinction in light emission wavelength. The discoveryhas led to another preferred set of acridinium-based chemiluminescentsubstrates, wherein the Formula VII is further defined as above, exceptthe substituents of R_(3c) can be any electron-donating group such ashydroxy, thiol, amino, monoalkylamino, dialkylamino, alkoxy orthioalkyl. The presence of this second electron-donating group furtherdecreases the energy gap between the electronically-excited and groundstates of the light-emitting acridone. Consequently, a further red-shiftin light emission is observed compared to 2-hydroxy-DMAE. This shiftalso results in increased spectral distinction of the blue andred-emitting acridinium ester pair.

One of the modes of enzyme detection disclosed in this invention is todetect long wavelength signal of the product (2) that arises fromenzymatic action. The commonly used methods for light detection includethe use of a luminometer, a CCD camera, X-ray film, and a high speedphotographic film. Since the hydrolytic enzyme to be detected oftenexists in a small quantity in the sample, the amount of the product thatis generated by the enzyme is relatively small in comparison with theamount of the substrate used in the reaction. Therefore, it is criticalto be able to detect such a small amount of this signal in the presenceof a strong signal of the substrate.

One important aspect of this invention related to detecting a longwavelength emission signal of the product such as 2 produced by ahydrolytic enzyme is the use of a luminometer having a red-sensitivephotomultiplier tube (PMT). (Frequently, this type of PMT is cooledduring use in order to reduce system noise.) The most commonly usedcommercial photomultiplier tubes are made from low dark count bialkalimaterial. It has an excellent quantum yield in the short wavelengthregion but a very low quantum yield in the long wavelength region. Forexample, bialkali PMT model R268 from Hamamatsu has a quantum yield ofabout 22% in the range of 400-500 nm and only ˜2% at 600 nm and above,which is not desirable for this specific application. On the other hand,PMT made from multialkali material is relatively more red-sensitive thanthat made from bialkali material. For instance, multialkali PMT modelR2228 from Hamamatu has a quantum yield of 3˜5% over the region of 400to 500 nm, and a quantum yield of ˜5% around 600 nm, which is moredesirable for this application. The drawback of this PMT is a high darkcount, and therefore it has to operate at low temperatures in order tosuppress the dark count. FIGS. 3 and 4 show quantum efficiencies of PMTsof bialkali R268 and multialkali R2228P, respectively.

Another useful aspect of this invention related to detecting a longwavelength emission signal of the product is the use of a charge-coupleddevice (CCD) camera, particularly a thinned, back-illuminated cooledCCD. As shown in FIG. 5, said CCD has a ˜80% quantum efficiency at 400nm and ˜90% at 700 nm, thus capable of increasing the detectability oflong wavelength emission (>550 nm) signal by 10-20 times or more overR268.

Since both the substrates and products of the present invention arechemiluminescent, an essential aspect of this invention related todetecting a long wavelength emission signal of 2 is the use of afiltering device to block the short wavelength signal from 1. FIGS. 6and 7 show transmittances of Corion LL700 and LL650 long wave passfilters (Corion, Franklin, MA), respectively.

One of the advantages in using chemiluminescent acridinium substrateslike 1 to detect hydrolytic enzymes is that the products like 2generated by the enzyme can be accumulated without undergoingsignificant decomposition during the enzymatic reaction. FIGS. 8 and 9show the stability of 2-OH-DMAE (2) in aqueous medium at pHs 9 and 10.5and at different elevated temperatures.

The sensitivity relating to the detection of a long emission signal ofthe product generated by a hydrolytic enzyme is largely dependent on thesignal differentiation of the product (2) and the substrate (1). It istherefore desirable to reduce background emission of the substrate2-Phos-DMAE in order to obtain a sensitive system. The generation oflight from acridinium compounds is traditionally made by the treatmentwith acidic hydrogen peroxide solution first, followed by an alkalinesurfactant solution. The purpose of the acid is to convert thepseudo-form of acridinium compound to the quaternary form and thesurfactant aids in enhancing the quantum yield of the acridiniumcompound. Preferably, said acidic hydrogen peroxide solution containshydrogen peroxide at concentration of 0.001 to 5%, and nitric acid atconcentration of 0.001 to 1.0 N, and said alkaline surfactant solutioncontains sodium hydroxide at concentration of 0.01 to 1 N, and AQUARD(CTAB) at 0.01 to 1%. More preferably, said acidic hydrogen peroxidesolution contains 0.5% hydrogen peroxide and 0.1 N nitric acid, and saidalkaline surfactant solution contains 0.25 N sodium hydroxide and 0.5%AQUARD (CTAB).

An unexpected finding, to the advantage of detecting the long wavelengthemission signal from 2, is that under certain conditions thechemiluminescence from 1 is selectively and significantly suppressed,and thereby the overall signal differentiation of 2 over 1 is improved.Specifically, the chemiluminescence of 1 is significantly lowered if thesolution is treated with an alkaline solution first and then withhydrogen peroxide solution. More specifically, when a solution of 1 istreated with 0.25 N sodium hydroxide solution containing 0.5% AQUARD,followed by 0.5% hydrogen peroxide, the quantum yield of 1 is lowered bymore than 30 fold. In contrast, under the same condition the quantumyield of 2 is basically not affected. This results in an overallimprovement of the signal differentiation by 30 fold. Thus, to theadvantage of better distinction between the signals of certainchemiluminescent products and their substrates, preferably, saidalkaline solution contains sodium hydroxide at concentration of 0.01 to1.0 N, and AQUARD at concentration of 0.01 to 1%, and said hydrogenperoxide solution contains hydrogen peroxide at concentration of 0.001to 5%. More preferably, said alkaline solution contains 0.25 N sodiumhydroxide and 0.5% AQUARD, and said hydrogen peroxide solution contains0.5% hydrogen peroxide.

With one or more of the above approaches combined, several assay systemsbased on the principle of detecting the long emission signal generatedby the action of AP on 2-Phos-DMAE (1) are constructed, and disclosed inthe section of Examples. One example (Example 17) consists of theincubation of AP standards in a substrate solution containing 0.1 mM2-Phos-DMAE (1), 1 mM magnesium chloride in 100 mM, pH 9 Tris buffer at45° C. for 1 hour. The light outputs were measured on a luminometerequipped with a red-sensitive PMT (R2228P) and a long pass filter(LL700), which is placed in a 4° C. cold room. The sample was treatedfirst with 0.25 N sodium hydroxide solution containing 0.5% AQUARD,immediately followed by 0.5% hydrogen peroxide. As shown in FIG. 12, APis detected at a level below 1×10-18 mole. Another example under thesimilar condition is given in Example 16 and the result is shown inFIGS. 10 and 11.

Another important method for selectively reducing background of2-Phos-DMAE (1) is through a selective quenching. Selective quenching,herein and hereafter, refers to the use of one or more compounds orchemical moieties to selectively reduce chemiluminescence of thesubstrate such as 1 via the mechanism of energy transfer. Referring backto Scheme III, the energy of the excited acridone (donor) resulting fromthe chemiluminescent reaction of the acridinium compound can betransferred to an adjacent, non-fluorescent molecule (acceptor orquencher). This non-fluorescent molecule reverts to the ground state viaa non-radiative pathway to release the energy. The quantum yield of theacridinium compound is quenched or reduced as the result of thequenching. The effectiveness of the quenching depends on the distance,spectral overlap and transition dipole-dipole interaction of theacridinium compound and the quencher. First, the acridinium compound andquencher must be in a close vicinity, preferably at a distance less than10 nm in order to achieve 20˜100% quenching effect, since theeffectiveness of energy transfer is inversely proportional to the sixthpower of the distance between the donor and acceptor. Secondly, the UVabsorption spectrum of the quencher must overlap, at least partially,with the emission spectrum of the acridinium compound. Lastly, eventhough it is often not easy to control the spatial conformations of thetwo concerned molecules in order to obtain the effective transitiondipole-dipole interaction, a relatively free movement of two spatiallyclose molecules often fulfil this requirement.

There are in general two ways of quenching, intermolecular andintramolecular. Specifically related to this invention, theintermolecular quenching refers to when the quencher and acridiniumcompound coexist in the same solution, but the two molecules are notlinked together. The intramolecular quenching refers to when both thequencher and acridinium compound are covalently linked in one molecule.In the case of intermolecular quenching, the effectiveness of thequenching depends on the concentration of the quencher relative to theconcentration of acridinium compound. The concentration of the quencheror substrate or both must be in the range of millimolar in order toachieve effective quenching. However, for the intramolecular quenching,the effectiveness of the quenching is determined by a bonding distancebetween the two molecules, and that in turn can be warranted by theproper selection of the length of the tether that connects the two.

Another aspect closely related to the selection of a quencher is thatthe quencher should only selectively or preferentially quench light of2-Phos-DMAE (1) and have little or no effect on the light emission of2-OH-DMAE (2). To achieve this, the UV absorption spectrum of thequencher should have a maximum overlap with emission spectrum of 1 but aminimum overlap, or preferably no overlap at all, with emission spectrumof 2. The other criteria for an effective quencher for this applicationinclude a large molar extinction coefficient and adequate watersolubility. Examples of compounds that are suitable to serve asquenchers are given below, but are not limited to those listed.

It is an intention of this invention to provide, but not limiting to,one mechanism to selectively reduce the light emission of the substratevia selective quenching. It is understandable that any other approach ormechanism capable of selectively reducing the quantum yield of thechemiluminescent substrates will also fall in the scope of thisinvention.

The second mode of light detection involves detecting the short emissionsignal of the substrate (1) that diminishes as the result of the actionof enzymatic action. This mode of detection takes advantage of the highquantum yield of bialkali, blue-sensitive PMTs as discussed earlier. Inconnection with the use of a blue-sensitive PMT, a filtering device thatis capable of blocking a long emission signal must be also used in orderto block long-wavelength light emission of the product in the hydrolyticenzymatic reaction.

Belonging to Formula II, another important sub-class of chemiluminescentsubstrates related to this invention are substrates capable of “reverseemission”, where Lumi-M-P is capable of emitting long-wavelength lightwhile the product (Lumi-M) is capable of emitting short-wavelengthlight. One major advantage in this reverse emission mode is that theproduct is detected at a short emission signal with bialkali,blue-sensitive PMT fitted with a short wave pass filter, where quantumyield of the PMT is as high as 22%.

Chemiluminescent substrates capable of the reverse emission arerepresented by Formula II where M-P is replaced by Formula X:

As provided in Formula X, preferably M and P are defined in Formula II.ED is an electron-donating group, preferably an ionizable group, whichdonates an electron pair to the conjugated system and is selected fromhydroxyl, —OR, —NR′R″, thiol (—SH), —SR, and —CHWn where n=1 or 2, W isan electron withdrawing group including, but not limited to, nitro,nitroso, cyano (—CN), —CHO, —C(O)R, —N⁺RR′R″, —CO₂H, —CO₂R, —S(O)R,—SO₂R, —SO₂OR, —SO₂NHR, —SO₂NR′R″-SO₃H, or F, where R is defined inFormula II, R′ and R″ are hydrogen or low alkyl, and R, R′ and R″ canall be the same or different. ED is interchangeable with R₁₂ or R₁₅.

R₁₂, R₁₃, R₁₄ and R₁₅ may be identical or different, and are selectedfrom hydrogen, —R, hydroxyl, amino, halides, nitro, nitroso, sulfonate,sulfate, phosphonate, —CO₂H, cyano (—CN), —SCN, —OR, —SR, —SSR, —C(O)R,and —C(O)NHR, and R is defined in Formula II; alternatively, anyadjacent two groups of R₁₂ to R₁₅ can form one or more additional fusedhydrocarbon aromatic rings or heteroaromatic rings with or withoutsubstitutions, and the additional fused hydrocarbon aromatic rings andheteroaromatic rings include, but are not limited to, benzene,naphthlene, pyridine, thiophene, furan, and pyrrole, and so on.

Preferably, R₁₂, R₁₃, R₁₄ and R₁₅ are all hydrogen, and ED is hydroxy,and chemiluminescent substrates capable of reverse emission have FormulaXI:

Alternatively, the group represented by Formula X can be located at theC₃ position of acridinium nucleus, having Formula XII below:

More preferably, in Formula XI and XII, M is oxygen; P is a phosphorylgroup, —PO₃Na₂, where two sodium cations can be exchanged independentlywith hydrogen, potassium, magnesium, calcium, or other cationic ion(s)or group(s) for maintaining the eletroneutrality of the molecule;R_(2a-c) and R_(3a-d) are hydrogen, R₁ is methyl; X is oxygen, Z isomitted, and Y is a polysubstituted aryl moiety of Formula III where R₄and R₈ are methyl, R₅ and R₇ are hydrogen, and R₆ is carboxyl (—CO₂H).One of the more preferred chemiluminescent substrate capable of reverseemission has structure 13. It is readily converted by alkalinephosphatase to its keto form (14).

Another preferred chemiluminescent substrate capable of reverse emissionis the reduced form of compound 13,

which is shown as 15. Similar to 13, compound 15 is also readilyconverted by alkaline phosphatase to its keto form (16).

It has been unexpectedly discovered that both compounds 13 and 15 arecapable of emitting light at extremely long wavelength. FIG. 2M is theemission spectrum of compound 15 determined by FFFS, showing that itemits light at λmax 678 nm. FIG. 2N is the emission spectrum of compound16, which is the product of 15 after the treatment with AP. It showsthat 16 emits light at λmax 450 nm, giving a net 228 nm of hypsochromicshift from 15.

Hypsochromic shift of emission wavelength due to a number of factorsincluding solvent and chemical substitutional effects on the acridiniumcompounds has been disclosed in the co-pending WO 00/09487, which isincluded herein as reference. Distinguishably, one of the discoveries inthis invention which relates to a large hypsochromic shift of emissionwavelength from 13 to 14 and from 15 to 16 is due to the interruption ofelectronic conjugation between the acridinium nucleus and theelectron-rich aromatic side chain caused by the enzymaticdephosphorylation.

2. Substrates and Products Having Different Emission Kinetics

In addition to spectral distinction as a means of discriminatingchemiluminescent substrate from product, enzyme-mediated, alteration ofacridinium-ester flash-kinetics is also an effective method to monitorand determine the concentration of the enzyme. This methodology thusprovides an alternate ‘readout’ signal when the enzyme is used as thelabel in an assay. One approach to modulate acridinium esterlight-emission kinetics is to alter the electronic properties of asuitable functional group located on the phenol, as shown in Reaction E.During the chemiluminescent reaction of acridinium esters, cleavage ofthe phenolic ester is a prerequisite for formation of the dioxetanoneprecursor that is ultimately responsible for light emission. If thecleavage of this phenolic ester can be altered in a predictable way bymaking the phenol a better or poorer leaving group, then the kinetics oflight emission from the corresponding acridinium ester can be modulated.For instance, an electron-donating group located ortho or para to thephenolic hydroxy moiety will render the phenol electron-rich, hence apoor leaving group. This will lead to slow light emission. Conversion ofthe electron-donating group to an electron-withdrawing group willaccelerate cleavage of the phenolic ester and fast light emission willbe obtained. The conversion of an electron-donating to anelectron-withdrawing group or vice versa can be accomplishedenzymatically. For instance, it is well known that oxidative enzymessuch as peroxidases can oxidize electron-donating groups such as aminoto electron-withdrawing groups such as nitroso or nitro groups. On theother hand, hydrolytic enzymes can convert an electron-withdrawing orneutral functional group to an electron-donating group. In this case thekinetics of light emission from an acridinium ester is slowed down bythe enzyme label.

Acridinium esters containing a 4′-hydroxy group on the phenol exhibitslow kinetics in their light emission upon reaction with hydrogenperoxide in strong alkaline solution. This is primarily because thephenol in the acridinium ester containing the 4′-hydroxy group is a poorleaving group. Conversion of this hydroxy group to a phosphate esterattenuates somewhat the electron donating ability of the 4′-oxygensubstituent. Light emission from acridinium esters containing a4′-phospho substituent is relatively faster than their 4′-hydroxycounterparts. Within this category are a subgroup of chemiluminescentacridinium substrates that are capable of emitting light at slowerkinetics after the treatment with a hydrolytic enzyme, said subgroup ofchemiluminescent acridinium substrates having Formula XIII. Wherein, R₁,R_(2a-c), R_(3a-d), A⁻, M, and P are as defined in Formula II, R_(2d) isas defined for R_(2a), R_(2b), R_(3b), and R_(3d), and R₅ and R₇ aredefined in Formula III. R₁₆ and R₁₇, identical or different, are thegroups selected from hydrogen, methyl, alkyl with low molecular weight,and halides. Preferably, R₁₆ and R₁₇ are different and one of them ishydrogen. More preferably, both R₁₆ and R₁₇ are hydrogen.

One of the more preferred chemiluminescent acridinium substrates ofFormula XIII is compound 4′-Phos-AE (17). As shown in Reaction F, 17 isreadily converted by AP to the product 4′-OH-AE (18). It was found thatat short measuring times (0.5-0.3 s), 17 emitted light ˜190 times fasterthan 18. While theoretically, either the concentration of the substrateor the product can be measured to estimate enzyme activity, it was foundto be more convenient (for effective signal discrimination) to measurethe chemiluminescent activity of the substrate, 4′-Phos-AE (17). Thechemiluminescent response to the concentration of alkaline phosphataseusing 17 as the substrate is given in FIG. 13.

3. Light Emission Spectra

The light emission spectra of compounds 1˜12 and 15˜18 were determinedby a Fast Spectral Scanning System (FSSS) of Photo Research (a divisionof Kollmorgen Corp.) of Burbank, Calif., U.S.A. The experiment wascarried out in a dark room. Each compound was dissolved in acetonitrileor N,N-dimethylformamide. The resulting concentrate was diluted with thesame solvent to form the working solution, which upon flashing gave alight emission with an adequate intensity. A typical experiment utilized10˜100 μg or more of the sample in 500 μl of the solvent contained in a13×100 mm borosilicate test tube. The tube was placed on a test tuberack raised to a proper height. A piece of aluminum foil was placed onthe back of the tube to enhance the detectability of the emitted light.The FSSS optical head was placed in front of the tube at an approximatedistance of about 130 mm with its lens focused on the liquid in thetube. The sample solution was first treated with 0.35 ml of the FlashingReagent #1 (Bayer Diagnostics) containing 0.1 N HNO₃ and 0.5% H₂O₂. Theroom was then darkened, and 0.35 ml of the Flashing Reagent #2 (BayerDiagnostics) containing 0.25 N NaOH and 0.5% ARQUAD was added to thereaction mixture immediately. (See U.S. Pat. No. 4,927,769, which iscommonly assigned and incorporated herein by reference.) The light whichwas generated instantaneously following the addition of the Reagent #2was recorded by FSSS for 5 seconds starting from about one second beforethe Reagent #2 was added. The various emission spectra determined onFSSS are given in FIGS. 2A-2P, and also summarized in Table 1. TABLE 1Emission Compound Range* (nm) Maximum (nm) 1 430-600 478 2500-780{circumflex over ( )} 602 3 430-600 474 4 500-780{circumflex over( )} 604 5 430-600 478 6 500-780{circumflex over ( )} 600 7 430-600 4748 500-780{circumflex over ( )} 594 9 440-600 476 10 520-780{circumflexover ( )} 674 11 430-600 474 12 510-780{circumflex over ( )} 638 15520-780{circumflex over ( )} 678 16 410-580 450 17 410-540 436 18410-540 452*Range is set for spectral region with signal intensity of above 5% ofpeak height.{circumflex over ( )}Emission spectral range goes beyond the scanninglimit (380-780 nm) of FSSS.4. Applications of the Chemiluminescent Enzyme Substrates in BindingAssays

While the specific example of the actual diagnostic assay disclosed hereuses 2-Phos-DMAE (1) as a chemiluminescent substrate for alkalinephosphatase, where alkaline phosphatase serves as a label for thedetection of human thyroid stimulating hormone (TSH) in serum, it isreasonable to conclude that a variety of hydrolytic enzymes could beused in conjunction with the appropriate chemiluminescent substrates ina variety of assay architectures for detection of either endogenous,diagnostic enzyme markers or other clinically relevant diagnosticmarkers should the hydrolase be used as a label. Therefore, while weclaim those assay architectures as should be obvious or otherwiseapparent to those who are skilled in the art of enzymatic, diagnosticassay, we do not restrict our claims to the following descriptions.

a. Conversion of Assay Readout Systems from Colorimetric or FluorescenceDetection to Chemiluminescence Detection:

The co-application of enzyme labels and their dependent luminescentchemistries to bioanalytical techniques has been reported as one ofseveral strategies for developing ultrasensitive clinical detectionmethodologies (Clin. Biochem., 26, 325, (1993)). Conversion of existingdiagnostic test formats from their current methods of calorimetric orfluorometric measurement to the potentially more sensitive method ofchemiluminescence detection would have a diverse multiplicity ofapplication in clinical analysis, especially since these calorimetric orfluorometric assays already exist in multitudinous configurations. Asmentioned earlier, chromogenic and fluorometric indicator substanceshave been used in sensitive immunoassays in the form of enzymesubstrates for detection of enzymes or the analytes to which theseenzymes are directed as labels. The present invention discloses ananalytical procedure where in general the chemiluminescent-emissionproperties of an enzyme substrate are distinguishable from the resultantproduct and that the enzymatic source for the catalytic conversion ofsaid substrate to product can be used to measure the enzyme activitydirectly for measurement of either the enzyme or the analytes to whichthe enzyme would be attached as a label.

b. An analyte, as in the example below on human TSH, is complexedspecifically with two different antibodies immobilized on solid phaseand tagged with an enzyme label, respectively. The quantity of analytein a sample should correlate to the quantity of captured label, which inthis case is alkaline phosphatase, and should ultimately correlate tothe observed magnitude of chemiluminescence. However, we envision morebroadly the following scheme of assay architectures generally dividedinto the two classes referred to independently as immunoassays andnucleic acid assays. We further divide these classes into eitherhomogeneous or heterogeneous assay configurations dependent on theseparation of bound from free analyte. Furthermore, we do not restrictour claims to those assay systems currently employing enzymes as labels,i.e., EIA, ELISA, Emit®, etc., but encompass also the realm of clinicaldiagnostics for which enzymes may be freely substituted for non-enzymelabels, i.e., radioisotopes, chromaphores, fluores, etc.

c. Heterogeneous Chemiluminescent Assay

An enzyme captured by a solid phase matrix, for example, a polystyrenesurface, magnetic particle, or combination of both, or precipitatingprotein, etc., either non-specifically, as with adsorption, orspecifically as through the attachment to the solid phase,non-covalently or otherwise, of a (directed) binding partner, includingcomplementary nucleic acid sequence, biotin, antibody, binding protein,receptor, ligand, etc. would be separated from interfering substancesthrough the separation of the solid phase from other assay componentsprior to application of the chemiluminescent substrate.

The enzyme could be either the endogenous diagnostic marker of interest,a label attached, covalently or otherwise, to a specific binding partner(collectively termed a tracer or probe) which is captured by the solidphase or a secondary reagent required for signal generation oramplification as in enzyme-cycling assays.

Examples of these assay configurations are widely reported in theliterature and compendiums of specific constructs have been published(Maggio, E., Enzyme-immunoassay, (1990) CRC Press), and (Wild, D., TheImmunoassay Handbook, (1994) Stockton Press).

A heterogeneous enzyme-immunoassay for the detection of an enzymephosphatase might be set up in several configurations. The simplestmethod would be to capture the phosphatase using an antibody which wouldspecifically bind the phosphatase to separable solid phase, washinginterfering substances from the solid phase prior to the addition ofchemiluminescent phosphorylated substrate. A second method directedtoward a protein analyte, could be fashioned in which a sandwich assayutilizing two antibodies, the first of which would be linked to a solidphase as described above, the second would be coupled to the signalgenerating system, alkaline phosphatase, β-galactosidase etc. Theconcentration of the enzyme label in the assay is then measured byapplication of the specific chemiluminescent substrate. Alternatively,in a competitive assay for a small analyte, a chemiluminescencegenerating enzyme such as alkaline phosphatase can be coupled to thesame hapten for use in the assay as a tracer, where an endogenousanalyte and the hapten-enzyme conjugate would compete for a limitednumber of solid phase binding sites using a similar signal readoutmechanism as is described above for the sandwich assay.

d. Homogeneous Chemiluminescent Assays

Assays that employ a homogeneous format can also be coupled to thepresent invention. Homogeneous assay architectures do not require theseparation of assay components to discriminate between negative andpositive analyte controls and therefore require fewer processing stepsthan heterogeneous assays. Homogeneous assay technologies such as Emit®and CEDIATM could be adapted to chemiluminescence detection. Emit®assays for small analytes could be developed in which both ahapten-enzyme conjugate, similar to that described above, and ananalyte-specific antibody would be added to a clinical sample. Theresultant chemiluminescence would be dependent on the degree of enzymeinhibition occurring with the formation of the antibody-enzyme complex.CEDIATM assays are envisioned in which a hapten is covalently conjugatedto the amino-terminal fragment of recombinant β-galactosidase or theenzyme donor (ED) fragment. Both the hapten-ED conjugate and an analytespecific antibody would then be incubated with the clinical sample inthe presence of the carboxy-terminal fragment of β-galactosidase, termedenzyme acceptor (EA) (Engel, W., Khanna, P., J. Immunol. Methods, 150,99 (1992)). The magnitude of resultant chemiluminescence from thesupposed enzyme substrate catalysis product, 2-hydroxy-DMAE, woulddepend on the degree of remaining enzyme inactivation.

e. Chemiluminescent Nucleic Acid Assays

We propose as obvious the application of said chemiluminescent enzymesubstrate systems for the labeling and/or detection of nucleic acids innucleic acid assays.

The invention disclosed herein is illustrated, but not limited, by thefollowing examples.

EXAMPLE 1

Synthesis of (2′,6′-dimethyl-4′-benzyl-oxycarbonyl)phenyl2-hydroxy-10-methyl-acridinium-9-carboxylate trifluoroacetate(2-OH-DMAE-Bn, 4).

4-Methoxyethoxymethoxy-iodobenzene

A solution of 4-iodophenol (10 g, 45.45 mmol) in 200 ml of anhydroustetrahydrofuran was treated at 0° C. with sodium hydride (2.36 g, 60%dispersion, 59.09 mmol) for 5 minutes. To the resulting mixture,methoxyethoxymethyl chloride (8.3 ml, 72.73 mmol) was slowly added overa 5-minute period. The mixture was stirred at 0° C. under nitrogen for30 minutes, warmed to room temperature, and stirred for 24 hours. Thesolvent was removed under reduced pressure. The residue was taken into500 ml of ether, washed with 5% sodium hydroxide (4×200 ml), water(4×200 ml), saturated sodium chloride (1×200 ml), and dried over sodiumsulfate. Evaporation of the solvent under reduced pressure gave an oilyproduct in 14.1 g. TLC (silica gel, ether): Rf 0.5.

N-(4-Methoxyethoxymethoxy)phenyl isatin

A solution of isatin (4.0 g, 27.2 mmol) in 200 ml of anhydrousN,N-dimethylformaldehyde was treated at room temperature with sodiumhydride (1.036 g, 60% dispersion, 32.64 mmol) for 0.5 hour, followed byaddition of 4-methoxyethoxymethoxy-iodobenzene (12.57 g, 40.8 mmol) andcopper (I) iodide (10.34 g, 54.4 mmol). The resulting mixture wasstirred at 160° C. under nitrogen for 17 hours. It was cooled to roomtemperature, and diluted with 400 ml of chloroform. The resultingmixture was filtrated to remove the inorganic materials. The filtratewas evaporated under reduced pressure to give a crude mixture containingN-(4-methoxyethoxymethoxy)phenyl isatin as a major product. TLC (silicagel, ether): Rf 0.8.

2-Methoxyethoxymethoxy-acridine-9-carboxylic acid

The above crude 4-methoxyethoxymethoxyphenyl isatin, withoutpurification, was suspended in 120 ml of 10% potassium hydroxide. Thesuspension was refluxed at 150° C. for 5 hours. After cooling to roomtemperature, the mixture was filtrated to remove the orange impurities.The filtrate was acidified in an ice-water bath with concentratedhydrochloric acid to pH 2. The resulting yellow precipitate wascollected and washed with water (4×50 ml) and air-dried. The driedmaterial was further washed with ether (6×50 ml) to yield the desiredproduct in 6.7 g. TLC (silica gel, 30% methanol/chloroform): Rf 0.5.

(2′,6′-Dimethyl-4′-benzyloxycarbonyl)phenyl2-methoxy-ethoxy-methoxy-acdidine-9-carboxylate

A suspension of 2-methoxyethoxymethoxy-acridine-9-carboxylic acid (3.6g, 11 mmol) in 150 ml of anhydrous pyridine was treated withp-toluenesulfonyl chloride (4.183 g, 22 mmol) at 0° C. for 5 minutes toform a homogeneous brown solution. Then, benzyl3,5-dimethyl-4-hydroxy-benzoate (2.818 g, 11 mmol) was added. Thesolution was stirred at room temperature under nitrogen for 20 hours.The solvent was removed under reduced pressure. The residue wasseparated on a silica flash chromatography column packed in hexane. Itwas eluted with 50% ether/hexane (1 liter) followed by 70% ether/hexane(3 liters). The product fraction was obtained from the 70% ether/hexaneeluent. Evaporation of the solvents under reduced pressure gave 3.74 gof the desired product. TLC (silica gel, ether): Rf 0.8.

(2′,6′-Dimethyl-4′-benzyloxycarbonyl)phenyl2-hydroxy-10-methyl-acridinium-9-carboxylate trifluoroacetate

A light-yellow solution of (2′,6′-dimethyl-4′-benzyloxycarbonyl)phenyl2-methoxyethoxymethoxy-acdidine-9-carboxylate (400 mg, 0.708 mmol) in 20ml of anhydrous methylene chloride was treated with methyltrifluoromethanesulfonate (0.4 ml, 3.54 mmol) at room temperature undernitrogen with stirring for 14 hours. The resulting mixture was treatedwith anhydrous ether (20 ml). The precipitate was collected and washedwith ether (4×20 ml) to yield 325 mg of the crude product. MS: (ESI):m/z 492.6 (M⁺). ¹H NMR (300 MHz, MeOD-d₄/CDCl₃): δ2.52 (6H, s), 5.01(3H, s), 5.42 (2H, s), 7.37-7.51 (5H, m), 7.81 (1H, d, J=2.6 Hz), 7.97(2H, s), 8.10 (1H, t, J=7.0 Hz), 8.16 (1H, dd, J₁=8.0 Hz, J₂=2.6 Hz),8.42 (1H, t, J=7.0 Hz), 8.60 (1H, d, J=8.8 Hz), and 8.73 (2H, twooverlapping doublets, J˜8.0 Hz). The product (25 mg) was furtherpurified on a preparative HLPC column (YMC, 250×30 mm, ODS, 10 μm),eluted in gradient by mixing 0.05% TFA/water (solvent A) and 0.05%TAF/acetonitrile (solvent B) in the following manner: 40 to 60% B in 40minutes, flow rate 20 ml/minute, monitored at 260 nm. The desiredproduct at retention time of ˜30 minutes was collected and crystallizedfrom methylene chloride/ether to give 17 mg of pure 4.

EXAMPLE 2

Synthesis of (2′,6′-dimethyl-4′-benzyloxy-carbonyl)phenyl2-phosphoryloxy-10-methyl-acridinium-9-carboxylate trifluoroacetate(2-Phos-DMAE-Bn, 3)

A solution of (2′,6′-dimethyl-4′-benzyloxycarbonyl)phenyl2-hydroxy-10-methyl-acridinium-9-carboxylate trifluoromethane-sulfonate(50 mg, 0.102 mmol) in pyridine (0.5 ml) was cooled to 0° C. and thenadded dropwise over a 3 minute period to a pre-cooled solution ofphosphorous oxychloride (57 μl, 6 eq.) in 0.5 ml of pyridine. Thereaction mixture was stirred at 0° C. under nitrogen for 30 minutes. Thereaction was quenched with 400 μl of 0.5 N NaOH followed by another 200μl of 1 N NaOH. The mixture was diluted with 1 ml of water, and thenfiltrated. The resulting yellow solid was dissolved in mixed DMF andwater, and separated on a preparative HPLC column (YMC, 250×30 mm, ODS,10 μm), eluted in gradient by mixing 0.05% TFA/water (solvent A) and0.05% TAF/acetonitrile (solvent B) in the following manner: 30 to 60% Bin 40 minutes, flow rate 20 ml/minute, monitored at 260 nm. The desiredproduct (3) at retention time of 32 minutes was obtained in 20 mg. MS(MALDI-TOF): m/z 573 (M+1).

EXAMPLE 3

Synthesis of (2′,6′-dimethyl-4′-carboxyl)phenyl2-hydroxy-10-methyl-acridinium-9-carboxylate trifluoroacetate(2-OH-DMAE, 2)

A solution of (2′,6′-dimethyl-4′-benzyloxy-carbonyl)phenyl2-hydroxy-10-methyl-acridinium-9-carboxylate trifluoromethanesulfonate(100 mg) in 4 ml of 30% hydrogen bromide in acetic acid was stirred at55° C. under nitrogen for 1 hour, and then treated with 10 ml ofanhydrous ether. The resulting precipitate was collected and washed withether (4×10 ml) to give 80 mg of (4-carboxyl-2,6-dimethyl)phenyl2-hydroxy-10-methyl-acridinium-9-carboxylate bromide. MS (ESI): m/z402.7 (M+). ¹H NMR (300 MHz, CD₃CN/MeOD-d₄): 52.52 (6H, s), 4.95 (3H,s), 7.78 (1H, d, J=2.7 Hz), 7.76 (2H, s), 8.10 (1H, t, J=7.0 Hz), 8.13(1H, dd, J₁=9.9 Hz, J₃₌₂=2.7 Hz), 8.40 (1H, d t, J₁=2.7 Hz, J₂=8.0 Hz),8.62 (1H, d, J=8.0 Hz), 8.77 (1H, d, J=9.2 Hz), and 8.78 (1H, d, J=9.9Hz). The further purification of the above product (68 mg) was carriedout by preparative HLPC (YMC, 250×30 mm, ODS, 10 μm), eluted in gradientby mixing 0.05% TFA/water (solvent A) and 0.05% TAF/acetonitrile(solvent B) in the following manner: 10 to 60% B in 40 minutes, flowrate 20 ml/minute, monitored at 260 nm. The desired product (2) atretention time of ˜27 minutes was obtained in 46 mg.

EXAMPLE 4

Synthesis of (2′,6′-dimethyl-4′-carboxyl)phenyl10-methyl-2-Phosphoryloxy-acridinium-9-carboxylate bromide (2-Phos-DMAE,1)

A mixture of (2′,6′-dimethyl-4′-benzyloxycarbonyl)phenyl10-methyl-2-phosphoryloxy-10-methyl-acridinium-9-carboxylatetrifluoroacetate (3, 37 mg) and 2.5 ml of 30% hydrogen bromide in aceticacid was stirred at 50° C. under nitrogen for 1.5 hour. The resultingmixture was cooled to room temperature, and treated with ether to formyellow precipitate. It was dissolved in mixed DMF/water with the help ofa small amount of triethylamine. The mixture was separated on apreparative HLPC column (YMC, 250×30 mm, ODS, 10 μm), eluted in gradientby mixing 0.05% TFA/water (solvent A) and 0.05% TAF/acetonitrile(solvent B) in the following manner: 10 to 60% B in 40 minutes, flowrate 20 ml/minute, monitored at 260 nm. The fraction at retention timeof ˜17 minutes was collected and lyophilized to dryness to give pure 1in 5.3 mg. MS (DALTI-TOF): m/z 482.818 (M+1).

EXAMPLE 5

Synthesis of phenyl 2-hydroxy-10-methyl-acridinium-9-carboxylatetrifluoromethanesulfonate (2-OH-AE, 6)

Phenyl 2-methoxyethoxymethoxy-acdidine-9-carboxylate

To a solution of 2-methoxyethoxymethoxy-acridine-9-carboxylic acid (101mg, 0.308 mmol) in 5 ml of anhydrous pyridine was addedp-toluenesulfonyl chloride (117 mg, 0.616 mmol). It was stirred at 0° C.for 5 minutes and at room temperature for an additional 10 minutesbefore 29 mg (0.038 mmol) of phenol was added. The reaction was stirredat room temperature under nitrogen overnight. The reaction mixture wasevaporated under reduced pressure. The residue was suspended in mixedmethanol and ethyl acetate and filtrated. The resulting filtrate wasreduced to a small volume and separated on 4 preparative silica gelplates (20×20 cm×2 mm thick), which were developed with hexane/ether(2:1). The major product band was collected and eluted with the samesolvent system. Removal of the solvents gave 27 mg of the pure product.TLC (silica gel, hexane/ether 2:1): Rf 0.9.

Phenyl 2-hydroxy-10-methyl-acridinium-9-carboxylatetri-fluoromethanesulfonate

A solution of phenyl 2-methoxyethoxymethoxy-acdidine-9-carboxylate (25mg, 0.062 mmol) in 1.5 ml of anhydrous methylene chloride was treatedwith methyl trifluoromethanesulfonate (38 μl, 0.336 mmol) at roomtemperature under nitrogen with stirring overnight. The reaction wasdiluted with another 0.5 ml of methylene chloride and then treated withanhydrous ether (4 ml). The resulting precipitate was collected andwashed with ether to give 6 in 19 mg. MS (ESI): m/z 330 (M⁺).

EXAMPLE 6

Synthesis of phenyl 10-methyl-2-Phosphoryloxy-acridinium-9-carboxylatetrifluoroacetate (2-Phos-AE, 5)

A solution of phenyl 2-hydroxy-10-methyl-acridinium-9-carboxylatetrifluoro-methanesulfonate (19 mg, 0.040 mmol) in pyridine (0.3 ml) wascooled to 0° C. and then added slowly to a pre-cooled solution ofphosphorous oxychloride (22 μl, 0.24 mmol) in 0.3 ml of pyridine. Thereaction mixture was stirred at 0° C. under nitrogen for 30 minutes, andquenched with 500 μl of 5% ammonium hydroxide for 10 minutes. Thesolution was then diluted with 1 ml of water and neutralized with 1 NHCl. The mixture was separated on a preparative HPLC column (YMC, 300×20mm, ODS, 10 μm), eluted in gradient by mixing 0.05% TFA/water (solventA) and 0.05% TAF/acetonitrile (solvent B) in the following manner: 10 to60% B in 40 minutes, flow rate 20 ml/minute, monitored at 260 nm. Thedesired product (5) at retention time of 18 minutes was obtained in 8mg. MS (ESI): m/z 410 (M⁺).

EXAMPLE 7

Synthesis of (2′, 6′-dimethyl-4′-carboxyl)phenyl2-phosphoryloxy-10-sulfobutyl-acridinium-9-carboxylate (2-Phos-NSB-DMAE,7)

(2′,6′-Dimethyl-4′-benzyloxycarbonyl)phenyl2-hydroxy-acridine-9-carboxylate

A solution of (2′,6′-dimethyl-4′-benzyloxy-carbonyl)phenyl2-methoxyethoxymethoxy-acridine-9-carboxylate (960 mg, 1.7 mmol) inmethylene chloride (5 ml) was treated with trifluoroacetic acid (5 ml)at room temperature for 19 hours. The reaction mixture was blown todryness with a nitrogen stream and the residue was suspended in ether.The product was collected and further washed with ether (2×10 ml) togive 610 mg. MS (MALTI-TOF): m/z 479 (M+1).

(2′,6′-Dimethyl-4′-benzyloxycarbonyl)phenyl2-dimethyl-phosphoryloxy-acridine-9-carboxylate

To a solution of (2′,6′-dimethyl-4′-benzyloxycarbonyl)phenyl2-hydroxy-acridine-9-carboxylate (50 mg, 0.105 mmol) in pyridine (2 ml)at 0° C. under nitrogen was added sodium hydride (5 mg, 0.208 mmol). Itwas allowed to stir at room temperature for 1 hour beforedimethoxychlorophosphate (56 μl, 0.519 mmol) was added. The reaction wascontinued to stir for 3 hours. The product was isolated from apreparative HLPC column (YMC, 250×30 mm, ODS, 10 μm). The column waseluted in gradient by mixing 0.05% TFA/water (solvent A) and 0.05%TFA/acetonitrile (solvent B) in the following manner: 40 to 80% B in 40minutes, flow rate at 20 ml/minute, monitored at 260 nm. The fraction atretention time of ˜52 minutes was collected and lyophilized to drynessto give the desired product in 45 mg. MS (MALTI-TOF): m/z 587 (M+1).

(2′,6′-Dimethyl-4′-benzyloxycarbonyl)phenyl2-dimethyl-phosphoryloxy-10-sulfobutyl-acridinium-9-carboxylate

A mixture of (2′,6′-dimethyl-4′-benzyloxycar-bonyl)phenyl2-dimethylphosphoryloxy-acridine-9-carboxylate (45 mg, 0.0768 mmol) and1,4-butanesultone (1 ml, 9.78 mmol) was stirred at 150° C. for 6 hoursunder nitrogen. The resulting thick gum was dissolved in a mixture ofacetonitrile/water. The solution was separated on a preparative HLPCcolumn (YMC, 250×30 mm, ODS, 10 μm). The column was eluted in gradientby mixing 0.05% TFA/water (solvent A) and 0.05% TFA/acetonitrile(solvent B) in the following manner: 40 to 80% B in 40 minutes, flowrate at 20 ml/minute, monitored at 260 nm. The fraction at retentiontime of ˜21 minutes was collected and lyophilized to dryness to give thedesired product in 1 mg. MS (MALTI-TOF): m/z 723 (M+1).

(2′,6′-Dimethyl-4′-carboxyl)phenyl2-phosphoryloxy-10-sulfo-butyl-acridinium-9-carboxylate

A solution of (2′,6′-dimethyl-4′-benzyl-oxycarbonyl)phenyl2-dimethylphosphoryloxy-10-sulfobutyl-acridinium-9-carboxylate (1 mg,0.00140 mmol) in chloroform (0.5 ml) was treated with boron tribromide(4 μl, 0.0423 mmol). It was stirred at room temperature under nitrogenfor 3 hours. The mixture was blown with a nitrogen stream to drynessthen dissolved in acetonitrile and water mixture. The desired productwas isolated from a semi-preparative HLPC column (Phenomenex, 300×7.8mm, ODS, 10 μm). The column was eluted in gradient by mixing. 0.05%TFA/water (solvent A) and 0.05% TFA/acetonitrile (solvent B) in thefollowing manner: 10 to 60% B in 40 minutes, flow rate at 2.5 ml/minute,monitored at 260 nm. The fraction at retention time of ˜17 minutes wascollected and lyophilized to dryness to give the desired product in 0.1mg. MS (MALTI-TOF): m/z 605 (M+1).

EXAMPLE 8

Synthesis of (2′,6′-dimethyl-4′-carboxyl)phenyl2-hydroxy-10-sulfobutyl-acridinium-9-carboxylate (2-OH-NSB-DMAE, 8)

N-(4-methoxyphenyl)isatin

A solution of isatin (2.5 g, 0.017 mol) in anhydrous DMF (50 ml) wascooled to 0° C. under a nitrogen atmosphere and treated with sodiumhydride (0.5 g, 1.2 equivalents). A purple solution was formed which wasstirred at 0° C. for 30 minutes and then warmed to room temperature.4-Bromoanisole (2.13 ml, 1 equivalent) was added followed by copperiodide (6.46 g, 2 equivalents). The reaction was heated in an oil-bathat 145° C. for 7 hours. The reaction was then cooled to room temperatureand diluted with an equal volume of ethyl acetate. This suspension wasfiltered and the filtrate was evaporated to dryness. TLC (1:4, ethylacetate:hexanes) indicated a very clean reaction; Rf (product)=0.5. Thecrude material was used as such for the next reaction.

2-Methoxy-acridine-9-carboxylic acid

The crude N-(4-methoxyphenyl)isatin from the above was suspended in 10%aqueous potassium hydroxide (150 ml) and refluxed under a nitrogenatmosphere. After 4 hours, the reflux was stopped and the reaction wasfiltered while still hot. The filtrate was diluted with water (˜150 ml)and ice. This solution was then acidified with concentrated hydrochloricacid. A yellow precipitate appeared which was collected by filtration.The precipitate was rinsed with cold water and ether and then air-dried.The dried residue was then transferred to a round-bottom flask with theaid of methanol and the partial solution was evaporated to dryness. Theresulting residue was evaporated to dryness from toluene twice. Ayellowish-brown powder was recovered in 1.15 g. TLC (1:4,methanol:chloroform) indicated a clean reaction; Rf (product)=0.14. Thismaterial was used as such for the next reaction.

2′,6′-Dimethyl-4′-benzyloxycarbonylphenyl2-methoxyacri-dine-9-carboxylate

2-Methoxyacridine-9-carboxylic acid (0.8 g, 0.0032 mol) in anhydrouspyridine (50 ml) was cooled in an ice-bath under a nitrogen atmosphereand treated with p-toluenesulfonyl chloride and4-benzyloxycarbonyl-2,6-dimethylphenol (0.81 g, 0.0032 mol). Thereaction was warmed to room temperature and stirred under a nitrogenatmosphere for 24 hours. The solvent was then removed under reducedpressure and the residue was dissolved in chloroform (10 ml). Theproduct was purified by flash chromatography using 5% ethyl acetate, 25%chloroform, 70% hexanes. Evaporation of the flash fractions containingthe product yielded a bright yellow solid in 0.84 g. MS (MALDI-TOF): m/zMS 492.8 (M+1)

(2′,6′-Dimethyl-4′-benzyloxycarbonyl)phenyl0.2-methoxy-10-sulfobutyl-acridinium-9-carboxylate

A mixture of (2′,6′-dimethyl-4′-benzyloxy-carbonyl)phenyl2-methoxy-acridine-9-carboxylate (200 mg, 0.407 mmol) in 2 ml of1,4-butanesultone was stirred at 150° C. for 19 hours under nitrogen.The resulting thick gum was dissolved in a mixed acetonitrile/watersolvent. The solution was separated on a preparative HLPC column (YMC,250×30 mm, ODS, 10 μm). The column was eluted in gradient by mixing0.05% TFA/water (solvent A) and 0.05% TFA/acetonitrile (solvent B) inthe following manner: 30 to 60% B in 40 minutes, flow rate at 20ml/minute, monitored at 260 nm. The fraction at retention time of ˜37minutes was collected and lyophilized to dryness to give the desiredproduct in 58.5 mg. MS (MALTI-TOF): m/z 629 (M+1).

(2′,6′-Dimethyl-4′-carboxyl)phenyl2-hydroxy-10-sulfobutyl-acridinium-9-carboxylate

A solution of (2′,6′-dimethyl-4′-benzyl-oxycarbonyl)phenyl2-methoxy-10-sulfobutyl-acridinium-9-carboxylate (50 mg, 0.080 mmol) inchloroform (4 ml) was treated with boron tribromide (50 μl, 0.529 mmol)and was stirred at room temperature under nitrogen for 3 hours. Thereaction was blown with a nitrogen stream to dryness then dissolved inacetonitrile and water mixture. The desired product was isolated from apreparative HLPC column (YMC, 250×30 mm I.D., ODS, 10 μm). The columnwas eluted in gradient by mixing 0.05% TFA/water (solvent A) and 0.05%TFA/acetonitrile (solvent B) in the following manner: 10 to 60% B in 40minutes, flow rate at 20 ml/minute, monitored at 260 nm. The fraction atretention time of ˜26 minutes was collected and lyophilized to drynessto give the desired product in 18 mg. MS (MALTI-TOF): m/z 525 (M+1).This compound was further purified, using another preparative HLPCcolumn (YMC, 300×20 mm I.D., ODS, 10 μm). The column was eluted ingradient by mixing 0.05% TFA/water (solvent A) and 0.05%TFA/acetonitrile (solvent B) in the following manner: 10 to 60% B in 40minutes, flow rate at 16 ml/minute, monitored at 260 nm. The pureproduct was obtained in 4 mg. MS (MALTI-TOF): m/z 525 (M+1).

EXAMPLE 9

Syntheses of (2′,6′-dimethyl-4′-carboxyl)phenyl2-hydroxy-7-methoxy-10-methyl acridinium-9-carboxylate (2-OH-7-MeO-DMAE,10) and (2′,6′-dimethyl-4′-carboxyl)phenyl2-phosphoryloxy-7-methoxy-10-methyl acridinium-9-carboxylate(2-Phos-7-MeO-DMAE, 9)

Synthesis of 4-benzyloxybromobenzene

4-Bromophenol (2 g, 0.0116 mol) in acetone (40 ml) was treated withpotassium carbonate (1.91 g, 1.2 equivalents) and benzyl bromide (1.44ml, 1.05 equivalents). The reaction was refluxed under nitrogen. After5-6 hours of reflux, the reaction was cooled to room temperature anddiluted with an equal volume of ethyl acetate. This was diluted furtherwith water and the organic layer was separated, dried over magnesiumsulfate and evaporated to dryness to afford a white fluffy powder.Yield=2.36 g (73%).

Synthesis of N-(4′-benzyloxy)phenyl-5-methoxyisatin

5-Methoxyisatin (1.5 g, 0.847 mmol) in anhydrous DMF (50 ml) was cooledin an ice-bath under nitrogen and treated with sodium hydride (0.25 g,1.2 equivalents). After 15-20 minutes in ice, a solution of4-benzyloxybromobenzene (2.36 g) in DMF (˜3 ml) was added along with CuI(3.23 g, 2 equivalents). The resulting reaction was heated in anoil-bath at 140° C. under nitrogen for 24 hours. The reaction was thenfiltered and the filtrate was evaporated to dryness. The residue wassuspended in ethyl acetate and purified by flash chromatography using35% ethyl acetate in hexane. Evaporation of the flash fractions affordedthe alkylated isatin as an orange-brown solid. Yield=1 g (32%).

Synthesis of 2-benzyloxy-7-methoxyacridine-9-carboxylic acid

The N-alkylated isatin from above (1 g) was suspended in 10% potassiumhydroxide (100 ml and refluxed under nitrogen for 4 hours. The reactionwas then filtered while still hot and the filtrate was cooled in ice.This was acidified carefully with a mixture of ice and concentrated HCluntil a thick yellow precipitate separated out. The precipitate wasallowed to stand for ˜15 minutes and was then collected by filtration.After rinsing with ether, the product was thoroughly air dried.Yield=0.75 g (75%).

Synthesis of (2′,6′-dimethyl-4′-benzyloxycarbonyl)phenyl2-benzyloxy-7-methoxy-acridine-9-carboxylate

2-Benzyloxy-7-methoxy-acridine-9-carboxylic acid (0.36 g, 0.001 mol) inanhydrous pyridine (30 ml) was cooled in an ice-bath under nitrogen andtreated with p-toluenesulfonyl chloride (0.39 g, 2 equivalents) followedby 4-carbobenzyloxy-2,6-dimethylphenol (0.3 g, 1.2 equivalents). Thereaction was warmed to room temperature and stirred for 16 hours undernitrogen. The solvent was then removed under reduced pressure and theresidue was dissolved in chloroform (˜60 ml). The chloroform solutionwas washed with 3% aqueous ammonium chloride. It was then dried overmagnesium sulfate and evaporated to dryness. The crude product waspurified by preparative TLC using 70% hexane, 27% chloroform, 3%methanol and isolated as a yellow solid. Yield=0.31 g (50%). MALDI-TOFMS 599.02 obs. (597.67 calc.).

Synthesis of (2′,6′-dimethyl-4′-benzyloxycarbonyl)phenyl2-benzyloxy-7-methoxy-10-methyl-acridinium-9-carboxylatetrifluoromethanesulfonate

The acridine ester from above (0.31 g, 0.52 mmol) was dissolved indichloromethane (5 ml) and treated with methyl trifluoromethanesulfonate(0.575 ml, 10 equivalents). The reaction was stirred at room temperaturefor 16 hours. Ether (150 ml) was then added and the precipitated productwas collected by filtration and air dried. Yield=0.23 g. MALDI-TOF MS613.33 obs. (612.7 calc.).

Synthesis of (2′,6′-dimethyl-4′-carboxyl)phenyl2-hydroxy-7-methoxy-10methyl-acridinium-9-carboxylate (10)

The acridinium ester from above (0.124 g) was stirred in a mixture ofmethyl sulfide and 30% HBr/AcOH (1:1, 4 ml). After 4 hours, ether wasadded to precipitate the product which was collected by filtration andair dried. This was dissolved in methanol and analyzed by analyticalHPLC using a 3.9×300 mm c18 column and a 30 minute gradient of 10-100%acetonitrile/water each containing 0.05% TFA at a flow rate of 1 ml/minand UV detection at 260 nm. The product was found to elute at 15 minuteswhile the starting material eluted at 24 minutes. Approximately, 60% ofthe crude material was purified by preparative HPLC and the HPLCfractions were lyophilized to dryness. Yield=42 mg (80%). MALDI-TOF MS432.87 obs. (432.45 calc.).

Synthesis of (2′,6′-dimethyl-4′carboxyl)phenyl2-phos-phoryloxy-7-methoxy-10-methyl-acridinium-9-carboxylate (9)

Crude deblocked acridinium ester from above (80 mg) was dissolved inpyridine (25 ml) and treated with phosphorus oxychloride (3×75 μl, ˜15equivalents) at 0° C. under nitrogen. The reaction was stirred for 1hour and then quenched with water (3 ml) and stirred for an additionalhour at room temperature. The reaction was then concentrated to a smallvolume. HPLC analysis using the same conditions as above but with a40-minute gradient of 10-60% acetonitrile/water (each with 0.05% TFA)showed product eluting at 20 minutes with starting material eluting at24 minutes. The product was isolated by preparative HPLC and the HPLCfractions were lyophilized to dryness. Yield=3 mg yellow powder.MALDI-TOF MS 513.00 obs. (513.26 calc.).

EXAMPLE 10

Synthesis of 2-OH-Spiroacridan (12) and 2-Phos-Spiroacridan (11)

N-(4′-Benzyloxy)phenylisatin

To a solution of isatin (4 g, 27.2 mmol) in DMF (50 ml) under nitrogenat room temperature was added NaH (871 mg, 34.5 mmol). The reactioncolor changed from orange to purple. It was stirred at room temperaturefor 30 min before 4-benzyloxyphenyl bromide (10.21 g, 38.8 mmol) and CuI(10.34 g, 54.4. mmol) were added. It was refluxed at 160° C. in anoil-bath for 20 hours under nitrogen. After cooling to room temperature,the reaction was poured into chloroform (400 ml), and filtrated. Thefiltrate was concentrated to dryness under reduced pressure to give thedesired product as a brown gum. It was used in the next step withoutfurther purification.

2-Benzyloxyacridine-9-carboxylic acid

The above mixture in 10% KOH/H₂O (220 ml) was refluxed at 130° C. for 20hours. The reaction was filtered while warm, and the filtrate was cooledto 0° C. before it was acidified with concentrated HCl to pH 3. Theyellow precipitate was filtered, and the filter cake was washed withwater (4×200 ml). It was dried under reduced pressure at 50° C. for 20hours. The desired product was obtained in 1.8 g. It was confirmed by MS(MALTI-TOF): m/z 331 (M+1).

(2′-Benzyloxy)phenyl 2-benzyloxyacridine-9-carboxylate

A solution of 2-benzyloxyacridine-9-carboxylic acid (306 mg, 0.93 mmol)in pyridine (30 ml) was treated with p-toluenesulfonyl chloride (276 mg,1.45 mmol) at room temperature under nitrogen for 30 min before2-(benzyloxy)phenol was added. The reaction was stirred at roomtemperature for 20 hours. It was concentrated to dryness under reducedpressure. The resulting material was purified on a flash column elutedwith gradient solvent system of ethyl acetate/hexane starting at 10%.The product came out at 20%. The desired fractions were combined andconcentrated to dryness under reduced pressure to give 226 mg of thedesired product. It was confirmed by MS (MALTI-TOF): m/z 513 (M+1).

(2′-Benzyloxy)phenyl 2-benzyloxy-10-methyl-acridine-9-carboxylatetrifluoromethanesulfonate

A solution of (2′-benzyloxy)phenyl 2-benzyloxyacridine-9-carboxylate(109 mg, 0.213 mmol) in dichloromethane (5 ml) was treated with methyltrifluoromethanesulfonate (260 μL, 2.30 mmol) under nitrogen at roomtemperature for 20 hours. The reaction was blown to dryness withnitrogen followed by suspending in ether. The yellow precipitate waswashed with more ether (4×10 ml). The resulting solid was dried underreduced pressure to give 71.35 mg of the desired product.

2-OH-Spiroacridan (11)

A mixture of (2′-benzyloxy)phenyl2-benzyloxy-10-methyl-acridine-9-carboxylate (30 mg, 0.043 mmol) in 30%HBr/AcOH (400 μL) was stirred at 45° C. for 1 hour. It was blown withnitrogen to dryness followed by suspending in ether. The solid wasfiltered, washed with more ether (4×10 ml), and dried under reducedpressure to give 17 mg of the desired product. A portion of this product(11 mg) was purified on a preparative HLPC column (YMC, 250×20 mm I.D.,ODS, 10 μm). The column was eluted in gradient by mixing 0.05% TFA/water(solvent A) and 0.05% TFA/acetonitrile (solvent B) in the followingmanner: 10 to 60% B in 40 minutes, flow rate at 16 ml/minute, monitoredat 260 nm. The fraction at retention time of ˜25 minutes was collectedand lyophilized to dryness to give the pure product in 7.3 mg. It wasconfirmed by MS (MALTI-TOF): m/z 347 (M+1).

2-Phos-Spiroacridan (12)

To a solution of the above 2-OH-Spiroacridan (5.66 mg, 0.016 mmol) inpyridine (0.5 ml) at 0° C. under nitrogen was added POC₃ (7.65 μl, 0.082mmol). It was stirred at the same temperature for 1 hour then quenchedwith water (0.5 ml) and stirred for another 15 min. It was concentratedto dryness under reduced pressure, followed by purification on apreparative HLPC column (YMC, 250×20 mm I.D., ODS, 10 μm). The columnwas eluted in gradient by mixing 0.05% TFA/water (solvent A) and 0.05%TFA/acetonitrile (solvent B) in the following manner: 10 to 60% B in 40minutes, flow rate at 16 ml/minute, monitored at 260 nm. The fraction atretention time of ˜18 minutes was collected and lyophilized to drynessto give the pure product in 2.3 mg. It was confirmed by MS (MALTI-TOF):m/z 427 (M+1).

EXAMPLE 11

Synthesis of (2′,6′-dimethyl-4′-carboxyl)phenyl3-(β-phosphoryloxy-4′-hydroxystyryl)-10-methyl acridinium-9-carboxylatetrifluoromethanesulfonate (3-Enol-Phos-DMAE, 13) and its correspondingacridan (3-Enol-Phos-acridan, 15)

Synthesis of N-[3-(1,3-dioxolyl)phenyl]isatin

Isatin (3.2 g, 0.0218 mol) was dissolved in anhydrous DMF (75 ml) andcooled in an ice-bath under a nitrogen atmosphere. To this coldsolution, sodium hydride (0.575 g, 0.0239 mol) was added and thereaction was stirred at 0° C. for 1.5 hours. This solution was thentreated with 2-(3-bromophenyl)-1,3-dioxolane (5 g, 1 equivalent)followed by CuI (8.3 g, 2 equivalents). The resulting suspension washeated in an oil-bath under nitrogen at 130-140° C. for 16 hours. It wasthen cooled to room temperature and diluted with an equal volume ofchloroform. This suspension was filtered and the filtrate wasconcentrated under reduced pressure. A viscous brown oil was recoveredwhich was suspended in xylenes (150 ml) wand evaporated to dryness. Theresidue was used as such for the next reaction. TLC (5% methanol inchloroform) showed clean conversion; Rf (product)=0.86.

Synthesis of 2-(9′-carboxyacridin-3′-yl)-1,3-dioxolane

Crude N-[3-(1,3-dioxolyl)phenyl]isatin from above was suspended in 10%KOH (150 ml) and the resulting suspension was refluxed under nitrogenfor 4.5 hours. The reaction was then cooled to room temperature andfiltered. The filtrate was diluted with ice and acidified with 20-30%HCl until weakly acidic. A yellow precipitate separated out which wascollected by filtration and air dried. Yield ˜5 g, yellow sticky solidwhich was used as such for the next reaction.

Synthesis of acridine-9-carboxylic acid-3-carboxaldehyde

Crude 2-(9′-carboxyacridin-3′-yl)-1,3-dioxolane (˜5 g) was suspended in80% aqueous acetic acid (100 ml). This suspension was heated at 80° C.under nitrogen for 16 hours. A yellow precipitate had appeared in thereaction. The reaction mixture was then cooled to room temperature anddiluted with anhydrous ether (˜500 ml). The precipitated solid wascollected by filtration, rinsed with ether and air dried. It was thentransferred to a round bottom flask, suspended in toluene (50 ml) andevaporated to dryness. This process was repeated once more. A brightyellow solid was recovered. Yield=1.72 g (31% overall). MALDI-TOF MS252.3 obs. (251.24 calc.).

Synthesis of (2′,6′-dimethyl-4′-benzyloxycarbonyl)phenylacridine-9-carboxylate-3-carboxaldehyde

Acridine-9-carboxylic acid-3-carboxaldehyde (0.3 g, 0.0012 mol) inpyridine (50 ml) was cooled in an ice-bath under nitrogen and treatedwith p-toluenesulfonyl chloride (0.456 g, 0.00239 mol). The reaction wasstirred at 0° C. for 15 minutes and then2,6-dimethyl-4-benzyoxycarbonyl-phenol (0.306 g, 1 equivalent) wasadded. The reaction was warmed to room temperature and stirred for 48hours under nitrogen and then concentrated under reduced pressure. Theresidue was dissolved in chloroform which was then washed with aqueousbicarbonate and aqueous ammonium chloride. The organic layer wasseparated, dried over magnesium sulfate and concentrated under reducedpressure. The crude residue (0.6 g) was purified by preparative TLC onsilica using 10% ethyl acetate in chloroform; Rf (product)=0.6.Yield=0.24 g (41%); bright yellow solid. MALDI-TOF MS 490.78 obs.(489.53 calc.); ¹H-NMR (CDCl₃): δppm 2.46 (s, 6H), 5.40 (s, 2H),7.37-7.50 (m, 5H), 7.78 (m, 1H), 7.94 (m, 3H), 8.12 (d, 1H, J=9.3 Hz),8.39 (d, 1H, J=8.6 Hz), 8.45 (d, 1H, J=8.6 Hz), 8.51 (d, 1H, J=9.2 Hz),8.79 (s, 1H), 10.31 (s, 1H).

Synthesis of (2′,6′-dimethyl-4′-benzyloxycarbonyl)phenyl3-[1′-hydroxy-2′-(4′-benzyoxyphenyl)ethyl]-acridine-9-carboxylate

Benzyloxybenzyl chloride (1 g, 0.0043 mol) in anhydrous THF (20 ml) wastreated with Mg turnings (˜0.5 g) which had been broken into smallerpieces to expose fresh surfaces. Upon slight warming, the Grignardreaction started and was cooled with cold water until the reaction wascomplete. The solution of the Grignard reagent was then cooled in a dryice-acetone bath and then added slowly via a syringe to a solution ofthe aldehyde from above (0.5 g, 0.001 mol) in THF (10 ml) which had beencooled thoroughly in a dry ice-acetone bath under nitrogen. The reactionwas stirred at −78° C. for 30 minutes by which time TLC showed completeconsumption of the starting material. The reaction was then diluted withethyl acetate (50 ml) and the resulting solution was poured into coldaqueous ammonium chloride (˜2%, 200 m). The organic layer was separated,dried over magnesium sulfate and evaporated to dryness. The cruderesidue was purified by preparative TLC on silica using 1:4 ethylacetate in hexane. The product was recovered as a yellow solid and wasused as such for the next reaction. Yield=0.38 g (54%).

Synthesis of (2′,6′-dimethyl-4′-benzyloxycarbonyl)phenyl3-(4′-benzyloxyphenylacetyl)-acridine-9-carboxylate

The alcohol from above (0.38 g, 0.55 mmol) in dichloromethane (30 ml)was treated with PCC (0.18 g, ˜5 equivalents). The reaction was stirredat room temperature under nitrogen. After 1 hour an additional 10equivalents PCC was added. After 2 hours, the reaction was diluted withethyl acetate (25 ml) and filtered. The filtrate was concentrated underreduced pressure. The residue was purified by preparative TLC on silicausing 5% ethyl acetate in chloroform. The product was recovered as abright yellow solid. Yield=80 mg (20%). ¹H-NMR (CDCl₃): δppm 2.46 (s,6H), 4.47 (s, 2H), 5.04 (s, 2H), 5.39 (s, 2H), 6.97 (d, 2H, J=8.7 Hz),7.26-7.49 (m, 10H), 7.76 (dd, 1H), 7.90 (dd, 1H), 7.95 (s, 2H), 8.23 (d,1H, J=9.3 Hz), 8.39 (d, 1H, J=8.7 Hz), 8.45 (d, 1H, J=6.9 Hz), 8.48 (d,1H, J=9.3 Hz), 9.02 (s, 1H). MALDI-TOF MS 687.27 obs. (686.78 calc.).

Synthesis of (2′,6′-dimethyl-4′-benzyloxycarbonyl)phenyl3-(β-dibenzylphosphotriester-4′-benzyloxystyryl)-acridine-9-carboxylate

Dibenzyl phosphite (0.263 g, 0.001 mol) in anhydrous benzene (4 ml) wastreated with N-chlorosuccinimide (0.134 g, 1 equivalent). The reactionwas stirred at room temperature under nitrogen for 1 hour. The acridineester ketone from above (80 mg, 0.117 mmol) was dissolved in anhydrousTHF (5 ml) and cooled to −78° C. under nitrogen in a dry ice-acetonebath and lithium bis(trimethylsilyl)amide (0.6-0.7 ml of 1.0 M) wasadded dropwise via a syringe. A purple solution was formed which wasstirred in the dry ice-acetone bath for 20 minutes and then the benzenesolution of dibenzylphosphochloridate was added dropwise and then warmedto room temperature. After ˜15 minutes, the dark purple color of thereaction faded to a dark yellow solution which was diluted with ethylacetate (25 ml), and then washed twice with aqueous ammonium chloride(˜2%). The organic layer was dried over magnesium sulfate and evaporatedto dryness. The product was purified by preparative TLC using 2:3 ethylacetate in hexane. Yield=59 mg (53%), orange-yellow oily solid. ¹H-NMR(CDCl₃): δppm 2.47 (s, 6H), 4.91 (m, 4H), 5.07 (s, 2H), 5.39 (s, 2H),6.76 (s, 1H, vinyl), 6.97 (d, 2H), 7.15-7.50 (m, 20H), 7.69 (m, 2H),7.87 (m, 1H), 7.96 (s, 2H), 8.34 (d, 1H), 8.41 (d, 1H), 8.57 (s, 1H).MALDI-TOF MS 947.11 obs. (947.01 calc.).

Synthesis of (2′,6′-dimethyl-4′-benzyloxycarbonyl)phenyl3-(β-dibenzylphosphotriester-4′-benzyloxystyryl)-10-methyl-acridinium-9-carboxylatetrifluoromethanesulfonate

The acridine enol-phosphate triester from above (59 mg, 0.0624 mmol) wasdissolved in dichloromethane (5 ml) and treated with methyltrifluoromethanesulfonate (71 μl, 10 equivalents). The reaction wasstirred at room temperature for 16 hours. HPLC analysis of the reactionmixture on a 3.9×300 mm, C18 column and a 30-minute gradient of 10-100%acetonitrile/water each containing 0.05% TFA at a flow rate of 1 ml/min.and UV detection at 260 nm; indicated product eluting at 19.2 minutes(60% conversion). The product was isolated by preparative HPLC and theHPLC fractions were concentrated under reduced pressure. The acridiniumester was isolated as a purple solid. Yield=29 mg (43%). MALDI-TOF MS962.11 obs. (962.04 calc.).

Synthesis of (2′,6′-dimethyl-4′-carboxyl)phenyl3-(β-phosphoryloxy-4′-hydroxystyryl)-10-methyl acridinium-9-carboxylatetrifluoromethanesulfonate (13)

The tetrabenzyl-protected acridinium ester from above (12.8 mg, 0.012mmol) was dissolved in methyl sulfide (1 ml) and treated with 30% HBr inacetic acid. The reaction was stirred at room temperature. After 4hours, ether (20 ml) was added and the precipitated solid was collectedby filtration. The product was dissolved in methanol (10 ml) andanalyzed by analytical HPLC (see above) which indicated clean conversionto product eluting at ˜16 minutes. The product was isolated bypreparative HPLC. The HPLC fractions were concentrated to a small volumeby rotary evaporation and then lyophilized to dryness. Yield=6 mg (71%).MALDI-TOF MS 601.34 obs. (600.54 calc.).

Synthesis of (2′,6′-dimethyl-4′-carboxyl)phenyl3-(β-phos-phoryloxy-4′-hydroxystyryl)-10-methyl acridan (15)

Crude tetrabenzyl-protected acridinium ester (˜40 mg) was dissolved in amixture of methanol (14 ml), acetone (5 ml) and 0.1 M ammonium acetatepH 6.9 (1.5 ml). This solution was treated with palladium black (18 mg).The resulting suspension was hydrogenated at room temperature using aballoon, and after 3-4 hours, a light green solution was obtained. Thereaction was then filtered and the filtrate was concentrated to a smallvolume and half the material was purified by preparative HPLC using a 25min. gradient of 30 to 90% methanol in water (each containing 0.05% TFA)on a C18 column (30×250 mm) and UV detection at 260 nm. The producteluted as a broad peak at ˜26.5 minutes. The HPLC fraction containingproduct was concentrated to dryness to afford a purple solid. Yield=8mg. MALDI-TOF MS 600.25 obs. (601.55 calc.).

EXAMPLE 12

Enzymatic conversion of (2′,6′-dimethyl-4′-carboxyl)phenyl3-(β-phosphoryloxy-4′-hydroxystyryl)-10-methyl acridinium-9-carboxylatetrifluoromethanesulfonate (13) to its keto product[3-(4′-Hydroxybenzyl)carbonyl-DMAE, 14]

A 1 mM DMF solution of the acridinium 3-enol-phosphate (13) was mixedwith 150 μL of 100 mM Tris pH 9 also containing 1 mM MgCl₂. Alkalinephosphatase (5 μl of a 2 mg/ml solution) was added and the reaction wasincubated at room temperature for ˜1 hour. HPLC analysis using a 3.9×300mm C18 column and a 30-minute gradient of MeCN in water, each containing0.05% TFA, at a flow rate of 1 ml/min and UV detection at 260 nm,indicated a complete conversion; Rt (starting material)=16 min, Rt(product)=20 min. The product was isolated by semi-preparative HPLCusing a 7.8×300 mm, C18 column. MALDI-TOF MS of the product indicatedthis was indeed the keto product (14): m/z 521.36 obs. (520.56 calc.).

EXAMPLE 13

Enzymatic conversion of (2′,6′-dimethyl-4′-carboxyl)phenyl3-(β-phosphoryloxy-4′-hydroxystyryl)-10-methyl acridan(3-Enol-Phos-acridan, 15) to its keto product[3-(4′-Hydroxybenzyl)carbonyl-acridan, 16]

A 20 μL DMF solution of the acridan (15) (1.7 mM) was combined with 180μl of 0.15 M 2-amino-2-methyl-1-propanol pH 10.3 also containing 1 mMmagnesium acetate. A clear blue solution was obtained (0.17 mMsubstrate), which was then treated with alkaline phosphatase (10⁻¹⁰moles). The blue color was discharged instantly. After one hour, HPLCanalysis using a C18 column (3.9×300 mm) and a 40-minute gradient offrom 10 to 90% MeCN/water each containing 0.05% TFA at a flow rate of 1mL/min and UV detection at 260 nm indicated a clean conversion to alater eluting product at 21.3 minutes (the starting material elutes at19.3 minutes). The reaction mixture was then evaluated by the FSSS todetermine the light emission spectrum of the product, which is shown inFIG. 2N. The product was isolated by semi-preparative HPLC. MALDI-TOF MSindicated that this was indeed the acridan keto compound (16): m/z521.67 obs. (521.56 calc.).

EXAMPLE 14

Synthesis of 4′-hydroxyphenyl 10-methyl acridinium-9-carboxylatetrifluoromethanesulfonate (4′-OH-AE, 18)

Synthesis of 1,4-dihydroxybenzene mono-tert-butyldimethyl-silyl ether

Hydroquinone (0.5 g, 0.0045 mol) in THF (25-30 ml) under nitrogen wastreated with imidazole (0.434 g, 1.5 equivalents) andtert-butyldimethylchlorosilane (0.686 g, 1 equivalent). The reaction wasstirred at room temperature under nitrogen. A precipitate appearedinstantly upon addition of the chlorosilane. After 3-4 hours, thereaction was diluted with ethyl acetate and washed with aqueous sodiumbicarbonate (˜2%) and brine. It was then dried over magnesium sulfateand evaporated to dryness. The product was purified by preparative TLCusing 25% ethyl acetate in hexanes. The product was obtained as an oil.Yield=0.5 g (50%).

Synthesis of 4′-tert-butyldimethylsilyloxyphenyl acridine-9-carboxylate

Acridine-9-carboxylic acid (0.29 g, 0.0013 mol) in anhydrous pyridine(10 ml) was cooled in ice under nitrogen and treated withp-toluenesulfonyl chloride (0.595 g, 2 equivalanets). After ˜10 minutesstirring in ice, hydroquinone mono-tertbutyldiemethylsilyl ether (0.29g) was added. The reaction was warmed to room temperature and stirredunder nitrogen for 16 hours. The reaction was then evaporated todryness. The residue was dissolved in ethyl acetate (50 ml) and washedwith aqueous sodium bicarbonate (˜2%). The ethyl acetate layer was driedover magnesium sulfate and evaporated to dryness. The crude product waspurified by preparative TLC on silica using 25% ethyl acetate inhexanes. Yield=0.25 g (45%). MALDI-TOF MS 431.61 obs. (429.58 calc.).

Synthesis of 4′-hydroxyphenyl 10-methyl acridinium-9-carboxylatetrifluoromethanesulfonate (18)

The acridine ester from above 90.25 g, 0.58 mmol) in dichloromethane(˜10 ml) was treated with methyl trifluoromethanesulfonate (0.66 ml, 10equivalents). The reaction was stirred at romm temperature for 16 hours.Ether was then added to precipitate the product which was collected byfiltration. A yellow powder was obtained. Yield=0.19 g. MALDI-TOF MS330.68 obs. (330.36 calc.).

EXAMPLE 15

Synthesis of 4′-phosphophenyl 10-methyl acridinium-9-carboxylatetrifluoromethanesulfonate (4′-Phos-AE, 17)

4′-Hydroxyphenyl 10-methyl acridinium-9-carboxylatetrifluoromethanesulfonate from above (32 mg, 0.069 mmol) was dissolvedin pyridine (1 ml) and cooled in an ice-bath under nitrogen. Phosphorusoxychloride (45 μl, 5 equivalents) was added and the reaction wasstirred in ice for 30 minutes. The reaction was then poured intoice-cold water (950 ml) containing 0.25 ml 5N NaOH. After stirringbriefly, this solution was extracted with chloroform and then ethylacetate. The aqueous solution was then lyophilized to dryness. A brightyellow powder was obtained which was analyzed by HPLC on a 3.9×300 mmC18 column and a 40 minute gradient of 10-90% acetonitrile/water, eachcontaining 0.05% TFA at a flow rate of 1 ml/min. and UV detection at 260nm. The product eluted at 12 minutes with very little starting materialat 15 minutes. The product was purified by preparative HPLC and the HPLCfraction was lyophilized to dryness. Yield=14 mg yellow, fluffy powder.MALDI-TOF MS 411.13 obs. (410.33 calc.).

EXAMPLE 16

Alkaline Phosphatase Assay Using 2-Phos-DMAE (1) (45° C., 0.5 hour)

The substrate solution of 2-Phos-DMAE (1) was prepared at 0.1 mM in 100mM, pH 9.0 Tris buffer containing 1 mM MgCl₂. The alkaline phosphatasestandards, Sigma Cat. No. P-3681, activity 4900 units (DEA)/mg protein,were prepared in water. Two μl each of alkaline phosphatase standardswas incubated with 48 μl of the substrate solution at 45° C. for 0.5hour, with 2 μL of water as a zero control. The solutions were thendiluted 100 fold with 100 mM, pH 9.0 Tris buffer containing 1 mM MgCl₂,respectively. Each diluent (25 μl) was flashed, in 5 replicates, with300 μl of 0.25 N NaOH containing 0.5% CTAB immediately followed by 300μl of 0.5% H₂O₂. The light outputs were measured for 2 seconds on BayerDiagnostics Magic Lite Analyzer 1 (MLA-1) equipped with R268 PMT and twoLL650 long wavelength pass filters (Corion, Lot No. CFS-002645). Theresult is shown in FIG. 10. The diluents were also flashed on MLA-1equipped with R2228P PMT and two LL650 long wavelength pass filters, andthe unit was pre-cooled in a cold room (4° C.). The result is given inFIG. 11.

EXAMPLE 17

Alkaline Phosphatase Assay Using 2-Phos-DMAE (1) (45° C., 1 hour)

According to Example 16, an assay of alkaline phosphatase was carriedout by adding 5 μl of the AP standard to 95 μl of the substratesolution. The solutions were incubated at 45° C. for 1 hour, and thendiluted 100 fold with the Tris buffe. The diluent (25 μl) was flashed in3 replicates with 300 μl of 0.25 N NaOH containing 0.5% CTAB immediatelyfollowed by 300 μl of 0.5% H₂O₂. The light outputs were measured for 2seconds on MLA-1 equipped with R2228P PMT and a LL700 long wavelengthpass filters, and the unit was pre-cooled in a cold room (4° C.). Theresult is given in FIG. 12.

EXAMPLE 18

Alkaline Phosphatase Assay Using 4′-phosphoryloxyphenyl-10-methylacridinium-9-carboxylate (4′-Phos-AE, 17)

Treatment of a solution of 4′-Phos-AE (17, 0.1 mM,) in 100 mM Tris pH 9,1 mM MgCl₂ with varying concentrations of alkaline phosphatase wascarried out. The reactions were incubated for 3-4 hours at roomtemperature and were then sequentially diluted 10⁴-fold into ‘flashbuffer’ which contains 10 mM phosphate pH 8 with 150 mM NaCl, 0.1% BSAand 0.05% sodium azide. Chemiluminescence of a 25 μL solution wasmeasured on a Bayer Diagnostics' Magic Lite Ananlyzer (MALL) equippedwith a BG38 filter. A measuring time of 0.3 s was employed. Evaluationof the chemiluminescent activity of the reactions indicated a steadydecrease in total chemiluminesence with increasing concentrations ofenzyme. Thus, as the concentration of enzyme is increased, a greaterproportion of the fast-emitting substrate 4′-Phos-AE is converted to theslow-emitting product 4′-OH-AE (18) leading to an overall decrease inlight output (at a short measuring time). The dose-response curve wasalso found to be sigmoidal in shape (a log-log plot is shown in FIG.13). Alkaline phosphatase was easily detected at 5×10⁻¹⁷ moles (0.1 mlreaction volume) in this assay.

EXAMPLE 19

Preparation of Anti-αTSH-Alkaline Phosphatase Conjugate

Thiolation of Murine, Monoclonal Anti-TSH

Murine, monoclonal antibody (32 nmole, 4.95 mgs) with binding affinityfor the α-subunit of human thyroid stimulating hormone (TSH) wasderivatized with 2-iminothiolane (0.32 pmole, 10 equivalents) in 0.10 MNa₂HPO₄, 0.15 M NaCl, 5.0 mM EDTA, pH 8.1 for 1 h. at ambienttemperature. The thiolated anti-αTSH was isolated in the void volume ofa Sephadex® G-25 fine column (1.5 cm i.d.×80 mLs) in 0.10 M Na₂HPO₄,0.15 M NaCl, 5.0 mM EDTA, pH 7.0. The yield was 4.65 mgs protein (93.4%recovery). Thiol incorporation as determined by MALDI-TOF MS was 3.8 peranti-αTSH.

Maleimide Activation of Alkaline Phosphatase

Calf intestinal alkaline phosphatase (EC 3.1.3.1) (39 nmole, 5.50 mgs)with a specific activity of 3,880 pNPP U/mg was derivatized withsulfosuccinimydyl 4-(N-maleimidomethyl) cyclohexane 1-carboxylate(sulfo-SMCC) (89 nmole, 2.3 equivalents) in 0.10 M Na₂HPO₄, 0.15 M NaCl,5.0 mM EDTA, pH 7.0 for 1 h. at ambient temperature. Themaleimido-alkaline phosphatase was isolated by retentive, centrifugalultrafiltration on a 30 kD molecular weight cutoff filter with multiplebuffer exchange cycles in 0.10 M Na₂HPO₄, 0.15 M NaCl, 5.0 mM EDTA, pH7.0. The yield was 4.64 mgs protein (84.4% recovery). Maleimideincorporation as determined by MALDI-TOF MS was 1.0 maleimide peralkaline phosphatase.

Conjugation of Maleimido-Alkaline Phosphatase to Thiolated Anti-αTSH

Maleimido-alkaline phosphatase (12 nmole, 0.62 equivalents) was coupledto thiolated anti-αTSH (19.4 nmole) in 0.10 M Na₂HPO₄, 0.15 M NaCl, 5.0mM EDTA, pH 7.0 for-16 h. at 4° C. The conjugate was isolated by SEC ona Sephadex® G-200 column (1.5 cm i.d.×125 mLs, 40-120 μm) in 0.10 MTris, 0.15 M NaCl, pH 7.4 at ambient temperature. Univalent conjugationwas confirmed by MALDI-TOF.

EXAMPLE 20

A Heterogeneous, Immunoassay Demonstrating 2-Phos-DMAE (1) Utility as aSubstrate for the Chemiluminescent Detection of TSH in Human Serum.

Diagnostic assay applicability of 2-Phos-DMAE as a chemiluminescentsubstrate was evaluated using a divalent, sandwich enzyme-immunoassay(EIA) formulated for the clinical quantitation of TSH in serum. In thisassay the alkaline phosphatase-anti-αTSH conjugate (henceforth referredto as a tracer) should bind specifically to the selected analyte, theα-subunit of intact human TSH, present in a patient sample or aTSH-containing standard (Bayer Diagnostics Corp., Walpole, Mass.), toform a noncovalently associating antibody-antigen complex. Thetracer-analyte complex is in turn captured by a magnetic bead solidphase, covalently coupled to a second, murine, monoclonal antibody withbinding affinity for the β-subunit of intact human TSH. Bound tracer ismagnetically separated from unbound tracer, and quantified by enzymatichydrolysis of the applied chemiluminescent substrate. Standard curvedata were used to calculate the TSH concentration of several controlsamples.

The tracer was diluted to a working concentration of 1.0 nM in ACS™ TSH3Lite Reagent Buffer (Bayer Diagnostics Corp., Walpole, Mass.). The TSHassay was initiated when 100 μl of the tracer were mixed with 200 μl ofeither a TSH standard or control. Eight TSH standards were used,containing TSH in concentrations of 0.000, 0.120, 0.740, 1.92, 3.86,8.99, 19.9, and 49.6 μI.U./ml (Bayer Diagnostics Corp., Walpole, Mass.).Three controls were also assayed. These were Ligands 1, 2 and 3 fromBayer Diagnostics, which contained TSH in mean concentrations of 0.60,5.1 and 18.4 μI.U./ml, respectively. The mixtures were collectivelyvortexed thrice for five seconds at setting number five on a Corning,Inc. model 4010 Multi-Tube Vortexer. Data points were acquired intriplicate. The assay mixtures were then incubated for thirty minutes atroom temperature, after which 225 μl of anti-βTSH MLP solid phase (˜56μgs) was added to each assay. The assay mixtures were vortexed thrice asdescribed above and incubated for thirty minutes at room temperature.The solid phase was magnetically separated from the supernatant by thethree-minute application of an array of permanent magnets in a BayerMagic Lite Assay Rack. The supernatant was decanted from the solidphase. Residual supernatant was removed by blotting for three minutesand then again for one minute. The solid phase was washed with twoseparate 1.0 ml volumes of water and suspended in 100 μl of substratesolution containing 0.10 mM 2-Phos-DMAE (1) in 100 mM Tris, 1.0 mMMgCl₂, pH 9.0. The enzyme reaction was carried out for 1 h. at 45° C.and was stopped with the addition of 2.0 ml of flashing buffercontaining 10 mM sodium phosphate, 0.15 M sodium chloride, 0.05% (w/v)sodium azide, 0.1% (w/v) BSA. The chemiluminescent reaction wasinitiated with the sequential addition of 300 μl each of Flash Reagent 1(0.25 N sodium hydroxide, 0.5% (w/v) N,N,N,N-hexadecyltrimethylammoniumchloride surfactant) and Flash Reagent 2 (0.5% (w/v) hydrogen peroxide)to 25 μl of the diluted reaction mixture on a Bayer Diagnostics MagicLite Analyzer equipped with two Corion LL-650 optical filters.Chemiluminescence data were collected as photons detected by the MagicLite Analyzer and expressed in relative light units (RLUs).

Method of Calculation for Sandwich Assay Parameters.

Arithmetic means for RLUs resulting from a specific analyteconcentration, represented here as μ, were calculated from threereplicates. Non-tracer assay reagents also contribute a small thoughsometimes significant number of RLUs. Hence, a control reaction,containing all assay reagents except tracer, was run in parallel todetermine non-tracer reagent background, represented here as n.Arithmetic mean RLUs, μ, were corrected to represent RLUs obtained fromthe tracer only, represented here as B, where B=μ−n. Where the analyteconcentration was highest, the corrected arithmetic mean RLU value forthat point was denoted as Bmax. A direct but non-linear relationshipexists between the analyte concentration present in the standard and thedetected RLUs. Consequently, the same direct sigmoidal correlation alsorelates the analyte concentration to the resultant % B/Bmax and may beaccurately expressed in the empirical linear form as$b = {{{- \log}\frac{y_{\infty} - y}{y - y_{0}}} - {m\quad\log\quad x}}$where x is the analyte concentration, and y is the observed signalgenerated either as % B/Bmax or RLUs (Ref. A, B and C). A. Rodbard,David, Ligand Analysis, (1981); and Langon, J., Clapp, J., (Eds.),Masson Publishing, Inc., New York, pp. 45-101; B. Nix, Barry, TheImmunoassay Handbook, (1994); and Wild, David (Ed.), Stockton Press,Inc., New York, pp. 117-123; and C. Peterman, Jeffrey H.,Immunochemistry of Solid-Phase Immunoassay; (1991); and Butler, J.(Ed.), CRC Press, Inc., Boca Raton; pp. 47-65.

Additionally, there are four more parameters, namely the regressionconstant, b, the regression coefficient, m, the assymptotic nonspecificbinding (NSB) at zero dose (analyte concentration), y_(∞), and theassymptotic infinite limit response for an infinitely high dose, y_(∞).The latter three of these parameters were calculated directly using theiterative, weighted, four-parameter logistic (4PL-WTD) analysis functionof the DOSECALC.EXE Rev.1.73 program (Bayer Diagnostics Corp., Walpole,Mass.). The arithmetic mean of the regression constant b was determinedover the entire range of analyte concentrations as calculated from thedose response expression re-written as$b = {{{- \log}\frac{y_{\infty} - y}{y - y_{0}}} - {m\quad\log\quad x}}$Analyte concentrations of unknowns were subsequently calculated usingthe dose response equation arranged as$x = {10\frac{{\log\left\lbrack {\left( {y_{\infty} - y} \right)/\left( {y - y_{0}} \right)} \right\rbrack} + b}{- m}}$

TSH enzyme-immunoassay standard curve using 2-Phos-DMAE (1) as achemiluminescent substrate. TSH assay data were plotted aschemiluminescence versus TSH concentration, which is shown in FIG. 14.Dynamic range extended for two orders of magnitude of analyteconcentration: satisfactory in this case for the accurate determinationof TSH concentration for the three control serum standards.

Assay accuracy in determination of TSH concentration. SH concentrationswere calculated for the Bayer Diagnostics Ligands 1, 2 and 3 using theweighted 4PL function. Calculated values closely matched the establishedvalues stated in the associated product literature. Therefore, thechemiluminescent substrate, 2-Phos-DMAE, has demonstrable utility forthe accurate determination of TSH concentration in human serum. Expectedvs. Calculated TSH Concentration for TSH Controls TSH ConcentrationChiron Diagnostics Ligands (μI.U./ml) 1 2 3 Expected 0.4-0.6 3.1-5.114.0-23.4 Range* Determined 0.5 3.9 16.8*Reported in Abbott IMx TSH kit (MEIA)

1. A chemiluminescent substrate of a hydrolytic enzyme, said substratehaving the structure

wherein P is PO₃B or a sugar moiety and B is a divalent cation or twomonovalent cations selected from the group consisting of Na₂, H₂, K₂, Caand Mg; M is oxygen; R₁ is an alkyl, alkenyl, alkynyl or aralkylcontaining 0 to 20 heteroatoms; R_(2a), R_(2b), R_(2c), R_(3a), R_(3b),R_(3c) and R_(3d) can be the same or different and are selected from thegroup consisting of hydrogen, methyl, methoxy, halide and cyano (—CN);R₁₂, R₁₃, R₁₄ and R₁₅ can be the same or different and are selected fromthe group consisting of hydrogen, —R, substituted or unsubstituted aryl,halides, nitro, sulfonate, sulfate, phosphonate, —CO₂H, —C(O)OR, cyano(—CN), —SCN, —OR, —SR, —SSR, —C(O)R, —C(O)NHR, ethylene glycol andpolyethyelene glycol, where R is an alkyl group having 1 to 6 carbonatoms; A⁻ is a counter ion for the electroneutrality of the quaternarynitrogen of the acridinium compound, said A⁻ not being present if saidR₁ substituent contains a strongly ionizable group that can form ananion and pair with the quaternary ammonium cationic moiety; and X isselected from the group consisting of O, N and S, such that, when X is Oor S, Y is selected from the group consisting of phenyl,(2′-methyl)phenyl, (2′-methoxy)phenyl, (2′,6′-dimethyl)phenyl,(2′,6′-dimethoxy)phenyl, (2′-methyl-6′-methoxy)phenyl,(2′,6′-dimethyl-4′-benzyloxycarbonyl)phenyl,(2′,6′-dimethoxy-4′-benzyloxycarbonyl)phenyl, (4′-methyl)phenyl,(2′-methyl-6′-methoxy-4′-benzyloxycarbonyl)phenyl,(2′,6′-dimethyl-4′-carboxyl)phenyl, (2′,6′-dimethoxy-4′-carboxyl)phenyland (2′-methyl-6′-methoxy-4′-carboxyl)phenyl, and Z is omitted, and whenX is nitrogen, Z is —SO₂—Y′, Y is as defined above, Y′ being defined thesame as Y above, or Y and Y′ can each be a branched or straight-chainalkyl containing 0 to 20 carbons, halogenated or unhalogenated, asubstituted aryl or heterocyclic ring system, and Y and Y′ can be thesame or different.
 2. The chemiluminescent substrate of claim 1 whereinany adjacent two groups of R₁₂, R₁₃, R₁₄ and R₁₅ can form one or moreadditional fused hydrocarbon aromatic rings or heteroaromatic rings withor without substitutions, said rings selected from the group consistingof benzene, naphthlene, pyridine, thiophene, furan and pyrrole.
 3. Thechemiluminescent substrate of claim 1 wherein said counter ion A⁻ isselected from the group consisting of CH₃SO₄ ⁻, FSO₃ ⁻, CF₃SO₃ ⁻,C₄F₉SO₃ ⁻, CH₃C₆H₄SO₃ ⁻, halide, CF₃COO⁻, CH₃COO⁻, and NO₃ ⁻.
 4. Thechemiluminescent substrate of claim 1 wherein R₁ is selected from thegroup consisting of methyl, carboxymethyl and sulfoalkyl.
 5. Thechemiluminescent substrate of claim 4 wherein R₁ is selected from thegroup consisting of sulfopropyl and sulfobutyl.
 6. The chemiluminescentsubstrate of claim 1 wherein P is PO₃B.
 7. The chemiluminescentsubstrate of claim 1 wherein R_(2a), R_(2b), R_(2c), R_(3a), R_(3b),R_(3c) and R_(3d) are hydrogen.
 8. The chemiluminescent substrate ofclaim 1 having the structure

wherein A⁻ is selected from the group consisting of CH₃SO₄ ⁻, FSO₃ ⁻,CF₃SO₃ ⁻, C₄F₉SO₃—, CH₃C₆H₄SO₃—, halide, CF₃COO⁻, CH₃COO⁻ and NO₃ ⁻. 9.The chemiluminescent substrate of claim 1 having the structure

wherein A⁻ is selected from the group consisting of CH₃SO₄ ⁻, FSO₃ ⁻,CF₃SO₃ ⁻, C₄F₉SO₃ ⁻, CH₃C₆H₄SO₃ ⁻, halide, CF₃COO⁻, CH₃COO⁻ and NO₃ ⁻.10. The chemiluminescent substrate of claim 1 having the structure

wherein A⁻ is selected from the group consisting of CH₃SO₄ ⁻, FSO₃ ⁻,CF₃SO₃ ⁻, C₄F₉SO₃ ⁻, CH₃C₆H₄SO₃ ⁻, halide, CF₃COO⁻, CH₃COO⁻ and NO₃ ⁻.11. The chemiluminescent substrate of claim 1 having the structure


12. The chemiluminescent substrate of claim 1 having the structure


13. A chemiluminescent substrate of a hydrolytic enzyme, said substratehaving the structure

wherein P is PO₃B or a sugar moiety and B is a divalent cation or twomonovalent cations selected from the group consisting of Na₂, H₂, K₂, Caand Mg; M is oxygen; R₁ is an alkyl, alkenyl, alkynyl or aralkylcontaining 0 to 20 heteroatoms; R_(2a), R_(2b), R_(2c), R_(2d), R_(3a),R_(3b), R_(3c) and R_(3d) can be the same or different and are selectedfrom the group consisting of hydrogen, methyl, methoxy, halide and cyano(—CN); R₅, R₇, R₁₆ and R₁₇ can be the same or different and are selectedfrom the group consisting of hydrogen, halides and —R, where R is analkyl group having 1 to 6 carbon atoms; and A⁻ is a counter ion for theelectroneutrality of the quaternary nitrogen of the acridinium compound,said A⁻ not being present if said R₁ substituent contains a stronglyionizable group that can form an anion and pair with the quaternaryammonium cationic moiety.
 14. The chemiluminescent substrate of claim 13wherein R₁ is selected from the group consisting of methyl,carboxymethyl and sulfoalkyl.
 15. The chemiluminescent substrate ofclaim 14 wherein R₁ is selected from the group consisting of sulfopropyland sulfobutyl.
 16. The chemiluminescent substrate of claim 13 whereinsaid counter ion A⁻ is selected from the group consisting of CH₃SO₄ ⁻,FSO₃ ⁻, CF₃SO₃ ⁻, C₄F₉SO₃ ⁻, CH₃C₆H₄SO₃ ⁻, halide, CF₃COO⁻, CH₃COO⁻, andNO₃ ⁻.
 17. The chemiluminescent substrate of claim 13 wherein R_(2a),R_(2b), R_(2c), R_(2d), R_(3a), R_(3b), R_(3c) and R_(3d) are hydrogen.18. The chemiluminescent substrate of claim 13 wherein R₁₆ and R₁₇ aredifferent and one of them is hydrogen.
 19. The chemiluminescentsubstrate of claim 13 wherein both R₁₆ and R₁₇ are hydrogen.
 20. Thechemiluminescent substrate of claim 13 wherein both R₁₆ and R₁₇ aremethyl.
 21. The chemiluminescent substrate of claim 13 having thestructure


22. The chemiluminescent substrate of claim 13 having the structure


23. The chemiluminescent substrate of claim 13 having the structure

wherein A⁻ is selected from the group consisting of CH₃SO₄, FSO₃ ⁻,CF₃SO₃ ⁻, C₄F₉SO₃ ⁻, CH₃C₆H₄SO₃ ⁻, halide, CF₃COO⁻, CH₃COO⁻ and NO₃ ⁻.